Immunoglobulins devoid of light chains

ABSTRACT

There is provided an isolated immunoglobulin comprising two heavy polypeptide chains sufficient for the formation of a complete antigen binding site or several antigen binding sites, wherein the immunoglobulin is further devoid of light polypeptide chains.

This is a division of application Ser. No. 08/106,944, filed Aug. 17,1993, now abandoned.

The invention relates to new isolated immunoglobulins which are devoidof light polypeptide chains. These immunoglobulins do not consist in thedegradation products of immunoglobulins composed of both heavypolypeptide and light polypeptide chains but to the contrary, theinvention defines a new member of the family of the immunoglobulins,especially a new type of molecules capable of being involved in theimmune recognition. Such immunoglobulins can be used for severalpurposes, especially for diagnosis or therapeutical purposes includingprotection against pathological agents or regulation of the expressionor activity of proteins.

Up to now the structure proposed for immunoglobulins consists of afour-chain model referring to the presence of two identical lightpolypeptide chains (light chains) and two identical heavy polypeptidechains (heavy chains) linked together by disulfide bonds to form a y- orT-shaped macromolecules. These chains are composed of a constant regionand a variable region, the constant region being subdivided in severaldomains. The two heavy polypeptide chains are usually linked bydisulphide bounds in a so-called "hinge region" situated between thefirst and second domains of the constant region.

Among the proteins forming the class of the immunoglobulins, most ofthem are antibodies and accordingly present an antigen binding site orseveral antigen binding sites.

According to the four-chain model, the antiger binding site of anantibody is located in the variable domains of each of the heavy andlight chains, and requires the association of the heavy and the lightchains variable domains.

For the definition of these four-chain model immunoglobulins, referenceis made to Roitt. I et al (Immunology-second-Edition Gower MedicalPublishing USA, 1989). Reference is especially made to the partconcerning the definition of the four-chain immunoglobulins, theirpolypeptidic and genetic structures, the definition of their variableand constant regions and the obtention of the fragments produced byenzymatic degradation according to well known techniques.

The inventors have surprisingly established that different molecules canbe isolated from animals which naturally produce them, which moleculeshave functional properties of immunoglobulins these functions being insome cases related to structural elements which are distinct from thoseinvolved in the function of four-chain immunoglobulins due for instanceto the absence of light chains.

The invention relates to two-chain model immunoglobulins which neithercorrespond to fragments obtained for instance by the degradation inparticular the enzymatic degradation of a natural four-chain modelimmunoglobulin, nor correspond to the expression in host cells, of DNAcoding for the constant or the variable region of a natural four-chainmodel immunoglobulin or a part of these regions, nor correspond toantibodies produced in lymphopaties for example in mice, rats or human.

E. S. Ward et al (1) have described some experiments performed onvariable domains of heavy polypeptide chains (V_(H)) or/and lightpolypeptide chains (V_(K) /F_(V)) to test the ability of these variabledomains, to bind specific antigens. For this purpose a library of V_(H)genes was prepared from the spleen genomic DNA of mice previouslyimmunized with these specific antigens.

Ward et al have described in their publication that V_(H) domains arerelatively sticky, presumably due to the exposed hydrophobic surfacenormally capped by the V_(K) or V.sub.λ domains. They consequentlyenvisage that it should be possible to design V_(H) domains havingimproved properties and further that V_(H) domains with bindingactivities could serve as the building blocks for making variablefragments (Fv fragments) or complete antibodies.

The invention does not start from the idea that the different fragments(light and heavy chains) and the different domains of these fragments offour-chain model immunoglobulin can be modified to define new orimproved antigen binding sites or a four-chain model immunoglobulin.

The inventors have determined that immunoglobulins can have a differentstructure than the known four-chain model and that such differentimmunoglobulins offer new means for the preparation of diagnosisreagents, therapeutical agents or any other reagent for use in researchor industrial purposes.

Thus the invention provides new immunoglobulins which are capable ofshowing functional properties of four-chain model immunoglobulinsalthough their structure appears to be more appropriate in manycircumstances for their use, their preparation and in some cases fortheir modification. Moreover these molecules can be considered as leadstructures for the modification of other immunoglobulins. The advantagewhich are provided by these immunoglobulins comprise the possibility toprepare them with an increased facility.

The invention accordingly relates to immunoglobulins characterized inthat they comprise two heavy polypeptide chains sufficient for theformation of a complete antigen binding site or several antigen bindingsites, these immunoglobulins being further devoid of light polypeptidechains. In a particular embodiment of the invention, theseimmunoglobulins are further characterized by the fact that they are theproduct of the expression in a prokaryotic or in a eukaryotic host cell,of a DNA or of a cDNA having the sequence of an immunoglobulin devoid oflight chains as obtainable from lymphocytes or other cells of Camelids.

The immunoglobulins of the invention can be obtained for example fromthe sequences which are described in FIG. 7.

The immunoglobulins of the invention, which are devoid of light chainsare such that the variable domains of their heavy chains have propertiesdiffering from those of the four-chain immunoglobulin V_(H). Thevariable domain of a heavy-chain immunoglobulin of the invention has nonormal interaction sites with the V_(L) or with the C_(H) 1 domain whichdo not exist in the heavy chain immunoglobulins. It is hence a novelfragment in many of its properties such as solubility and position ofthe binding site. For clarity reasons we will call it V_(HH) in thistext to distinguish it from the classical V_(H) of four-chainimmunoglobulins.

By "a complete antigen binding site" it is meant according to theinvention, a site which will alone allow the recognition and completebinding of an antigen. This could be verified by any known methodregarding the testing of the binding affinity.

These immunoglobulins which can be prepared by the technique ofrecombinant DNA, or isolated from animals, will be sometimes called"heavy-chain immunoglobulins" in the following pages. In a preferredembodiment of the invention, these immunoglobulins are in a pure form.

In a first embodiment, the immunoglobulins of the invention areobtainable in prokaryotic cells, especially in E. coli cells by aprocess comprising the steps of:

a) cloning in a Bluecript vector of a DNA or cDNA sequence coding forthe V_(HH) domain of an immunoglobulin devoid of light chain obtainablefor instance from lymphocytes of Camelids,

b) recovering the cloned fragment after amplification using a 5' primercontaining an Xho site and a 3' primer containing the Spe site havingthe following sequence

TC TTA ACT AGT GAG GAG ACG GTG ACC TG, SEQ ID NO: 51

c) cloning the recovered fragment in phase in the immuno PBS vectorafter digestion of the vector with Xho and Spe restriction enzymes,

d) transforming host cells, especially E. coli by transfection with therecombinant immuno PBS vector of step c,

e) recovering the expression product of the V_(HH) coding sequence, forinstance by using antibodies raised against the dromadary V_(HH) domain.

In another embodiment the immunoglobulins are hetero-specificimmunoglobulins obtainable by a process comprising the steps of:

obtaining a first DNA or cDNA sequence coding for a V_(HH) domain orpart thereof having a determined specificity against a given antigen andcomprised between Xho and Spe sites,

obtaining a second DNA or cDNA sequence coding for a V_(HH) domain orpart thereof, having a determined specificity different from thespecificity of the first DNA or cDNA sequence and comprised between theSpe and EcoRI sites,

digesting an immuno PBS vector with EcoRI and XhoI restriction enzymes,

ligating the obtained DNA or cDNA sequences coding for V_(HH) domains,so that the DNA or cDNA sequences are serially cloned in the vector,

transforming a host cell, especially E. coli cell by transfection, andrecovering the obtained immunoglobulins.

In another embodiment, the immunoglobulins are obtainable by a processcomprising the steps of:

obtaining a DNA or cDNA sequence coding for a V_(HH) domain or partthereof, having a determined specific antigen binding site,

amplifying the obtained DNA or cDNA, using a 5' primer containing aninitiation codon and a HindIII site, and a 3' primer containing atermination codon having a XhoI site,

recombining the amplified DNA or cDNA into the HindIII (position 2650)and XhoI (position 4067) sites of a plasmid pMM984,

transfecting permissive cells especially NB-E cells with the recombinantplasmid,

recovering the obtained products.

Successful expression can be verified with antibodies directed against aregion of a V_(HH) domain, especially by an ELISA assay.

According to another particular embodiment of this process, theimmunoglobulins are cloned in a parvovirus.

In another example these immunoglobulins are obtainable by a processcomprising the further cloning of a second DNA or cDNA sequence havinganother determined antigen binding site, in the pMM984 plasmid.

Such an Immunoglobulin can be further characterized in that it isobtainable by a process wherein the vector is Yep 52 and the transformedrecombinant cell is a yeast especially S. cerevisiae.

A particular Immunoglobulin is characterized in that it has a catalyticactivity, especially in that it is directed against an antigen mimickingan activated state of a given substrate. These catalytic antibodies canbe modified at the level of their biding site, by random or directedmutagenesis in order to increase oe modify their catalytic function.Reference may be made to the publication of Lerner et al (TIBS November1987. 427-430) for the general technique for the preparation of suchcatalytic immunoglobulins.

According to a preferred embodiment, the immunoglobulins of theinvention are characterized in that their variable regions contain inposition 45, an amino-acid which is different from leucine, proline orglutamine residue.

Moreover the heavy-chain immunoglobulins are not products characteristicof lymphocytes of animals nor from lymphocytes of a human patientsuffering from lymphopathies. Such immunoglobulins produced inlymphopathies are monoclonal in origin and result from pathogenicmutations at the genomic level. They have apparently no antigen bindingsite.

The two heavy polypeptide chains of these immunoglobulins can be linkedby a hinge region according to the definition of Roitt et al.

In a particular embodiment of the invention, immunoglobulinscorresponding to the above-defined molecules are capable of acting asantibodies.

The antigen binding site(s) of the immunoglobulins of the invention arelocated in the variable region of the heavy chain.

In a particular group of these immunoglobulins each heavy polypeptidechain contains one antigen binding site on its variable region, andthese sites correspond to the same amino-acid sequence.

In a further embodiment of the invention the immunoglobulins arecharacterized in that their heavy polypeptide chains contain a variableregion (V_(HH)) and a constant region (C_(H)) according to thedefinition of Roitt et al, but are devoid of the first domain of theirconstant region. This first domain of the constant region is calledC_(H) 1.

These immunoglobulins having no C_(H) 1 domain are such that thevariable region of their chains is directly linked to the hinge regionat the C-terminal part of the variable region.

The immunoglobulins of the type described hereabove can comprise type Gimmunoglobulins and especially immunoglobulins which are defined asimmunoglobulins of class 2 (IgG2) or immunoglobulins of class 3 (IgG3).

The absence of the light chain and of the first constant domain lead toa modification of the nomenclature of the immunoglobulin fragmentsobtained by enzymatic digestion, according to Roitt et al.

The terms Fc and pFc on the one hand, Fc' and pFc' on the other handcorresponding respectively to the papain and pepsin digestion fragmentsare maintained.

The terms Fab F(ab)₂ F(ab')₂ Fabc, Fd and Fv are no longer applicable intheir original sense as these fragments have either a light chain, thevariable part of the light chain or the C_(H) 1 domain.

The fragments obtained by papain digestion and composed of the V_(HH)domain and the hinge region will be called FV_(HH) h or F(V_(HH) h)₂depending upon whether or not they remain linked by the disulphidebonds.

In another embodiment of the invention, immunoglobulins replying to thehereabove given definitions can be originating from animals especiallyfrom animals of the camelid family. The inventors have found out thatthe heavy-chain immunoglobulins which are present in camelids are notassociated with a pathological situation which would induce theproduction of abnormal antibodies with respect to the four-chainimmunoglobulins. On the basis of a comparative study of old worldcamelids (Camelus bactrianus and Camelus dromaderius) and new worldcamelids (for example Lama paccos, Lama glama, and Lama vicugna) theinventors have shown that the immunoglobulins of the invention, whichare devoid of light polypeptide chains are found in all species.Nevertheless differences may be apparent in molecular weight of theseimmunoglobulins depending on the animals. Especially the molecularweight of a heavy chain contained in these immunoglobulins can be fromapproximately 43 kd to approximately 47 kd, in particular 45 kd.

Advantageously the heavy-chain immunoglobulins of the invention aresecreted in blood of camelids.

Immunoglobulins according to this particular embodiment of the inventionare obtainable by purification from serum of camelids and a process forthe purification is described in details in the examples. In the casewhere the immunoglobulins are obtained from Camelids, the inventionrelates to immunoglobulins which are not in their natural biologicalenvironment.

According to the invention immunoglobulin IgG2 as obtainable bypurification from the serum of camelids can be characterized in that:

it is not adsorbed by chromatography on Protein G Sepharose column,

it is adsorbed by chromatography on Protein A Sepharose column,

it has a molecular weight of around 100 kd after elution with a pH 4.5buffer (0.15 M NaCl, 0.58% acetic acid adjusted to pH 4.5 by NaOH),

it consists of heavy λ2 polypeptide chains of a molecular weight ofaround 46 kd preferably 45 after reduction.

According to a further embodiment of the invention another group ofimmunoglobulins corresponding to IgG3, as obtainable by purificationfrom the serum of Camelids is characterized in that the immunoglobulin:

is adsorbed by chromatography on a Protein A Sepharose column,

has a molecular weight of around 100 kd after elution with a pH 3.5buffer (0.15 M NaCl, 0.58% acetic acid),

is adsorbed by chromatography on a Protein G Sepharose column and elutedwith pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid).

consists of heavy γ3 polypeptide chains of a molecular weight of around45 Kd in particular between 43 and 47 kd after reduction.

The immunoglobulins of the invention which are devoid of light chains,nevertheless comprise on their heavy chains a constant region and avariable region. The constant region comprises different domains.

The variable region of immunoglobulins of the invention comprisesframeworks (FW) and complementarity determining regions (CDR),especially 4 frameworks and 3 complementarity regions. It isdistinguished from the four-chain immunoglobulins especially by the factthat this variable region can itself contain an antigen binding site orseveral, without contribution of the variable region of a light chainwhich is absent.

The amino-acid sequences of frameworks 1 and 4 comprise among othersrespectively amino-acid sequences which can be selected from thefollowing:

for the framework 1 domain

    G G S V Q T G G S L R L S C E I S G L T F D                                                                 SEQ ID NO:1                                        - G G S V Q T G G S L R L S C A V S G F S F S SEQ ID NO:2                     - G G S E Q G G G S L R L S C A I S G Y T Y G SEQ ID NO:3                     - G G S V Q P G G S L T L S C T V S G A T Y S SEQ ID NO:4                     - G G S V Q A G G S L R L S C T G S G F P Y S SEQ ID NO:5                     - G G S V Q A G G S L R L S C V A G F G T S SEQ ID NO:6                       - G G S V Q A G G S L R L S C V S F S P S S SEQ ID NO:7                       - for the framework 4 domain                                                  - W G Q G T Q V T V S S SEQ ID NO:8                                           - W G Q G T L V T V S S SEQ ID NO:9                                           - W G Q G A Q V T V S S SEQ ID NO:10                                          - W G Q G T Q V T A S S SEQ ID NO:11                                          - R G Q G T Q V T V S L SEQ ID NO:12                                          - for the CDR3 domain                                                         - A L Q P G G Y C G Y G X - - - - - - - - - - C L SEQ ID NO:62                                                     - V S L M D R I S Q H - - - - - -                                           - - - - - - G C SEQ ID NO:63                                                    - V P A H L G P G A I L D L K K Y                                           - - - - - - K Y SEQ ID NO:64                                                    - F C Y S T A G D G G S G E - - -                                           - - - - - - M Y SEQ ID NO:65                                                    - E L S G G S C E L P L L F - - -                                           - - - - - - D Y SEQ ID NO:66                                                    - D W K Y W T C G A Q T G G Y F -                                           - - - - - - G D SEQ ID NO:67                                                    - R L T E M G A C D A R W A T L A                                           T R T F A Y N Y SEQ ID NO:68                                                    - Q K K D R T R W A E P R E W - -                                           - - - - - - N N SEQ ID NO:69                                                    - G S R F S S P V G S T S R L E S                                           - S D Y - - N Y SEQ ID NO:70                                                    - A D P S I Y Y S I L X I E Y - -                                           - - - - - - K Y SEQ ID NO:71                                                    - D S P C Y M P T M P A P P I R D                                           S F G W - - D D SEQ ID NO:72                                                    - T S S F Y W Y C T T A P Y - - -                                           - - - - - - N V SEQ ID NO:73                                                    - T E I E W Y G C N L R T T F - -                                           - - - - - - T R SEQ ID NO:74                                                    - N Q L A G G W Y L D P N Y W L S                                           V G A Y - - A I SEQ ID NO:75                                                    - R L T E M G A C D A R W A T L A                                           T R T F A Y N Y SEQ ID NO:76                                                    - D G W T R K E G G I G L P W S V                                           Q C E D G Y N Y SEQ ID NO:77                                                    - D S Y P C H L L - - - - - - - -                                           - - - - - - D V SEQ ID NO:78                                                    - V E Y P I A D M C S - - - - - -                                           - - - - - - R Y SEQ ID NO:79        

As stated above, the immunoglobulins of the invention are preferablydevoid of the totality of their C_(H) 1 domain.

Such immunoglobulins comprise C_(H) 2 and C_(H) 3 domains in theC-terminal region with respect to the hinge region.

According to a particular embodiment of the invention the constantregion of the immunoglobulins comprises C_(H) 2 and C_(H) 3 domainscomprising an amino-acid sequence selected from the following:

for the C_(H) 2 domain:

    APELLGGPTVFIFPPKPKDVLSITLTP                                                                          SEQ ID NO:31                                              - APELPGGPSVFVFPTKPKDVLSISGRP SEQ ID NO:32                                    - APELPGGPSVFVFPPKPKDVLSISGRP SEQ ID NO:33                                    - APELLGGPSVFIFPPKPKDVLSISGRP SEQ ID NO:34                                    - for the C.sub.H 3 domain:                                                   - GQTREPQVYTLA SEQ ID NO:35                                                   - GQTREPQVYTLAPXRLEL SEQ ID NO:36                                             - GQPREPQVYTLPPSRDEL SEQ ID NO:109                                            - GQPREPQVYTLPPSREEM SEQ ID NO:110                                            - GQPREPQVYTLPPSQEEM SEQ ID NO:111                                     

Interestingly the inventors have shown that the hinge region of theimmunoglobulins of the invention can present variable lengths. Whenthese immunoglobulins act as antibodies, the length of the hinge regionwill participate to the determination of the distance separating theantigen binding sites.

Preferably an immunoglobulin according to the invention is characterizedin that its hinge region comprises from 0 to 50 amino-acids.

Particular sequences of hinge region of the immunoglobulins of theinvention are the following.

    GTNEVCKCPKCP            SEQ ID NO:37                                             - or,                                                                         - EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP SEQ ID NO:38                     

The short hinge region corresponds to an IgG3 molecule and the longhinge sequence corresponds to an IgG2 molecule.

Isolated V_(HH) derived from heavy chain immunoglobulins or V_(HH)libraries corresponding to the heavy chain immunoglobulins can bedistinguished from V_(HH) cloning of four-chain model immunoglobulins onthe basis of sequence features characterizing heavy chainimmunoglobulins.

The camel heavy--chain immunoglobulin V_(HH) region shows a number ofdifferences with the V_(HH) regions derived from 4-chain immunoglobulinsfrom all species examined. At the levels of the residues involved in theV_(HH) /V_(L) interactions, an important difference is noted at thelevel of position 45 (FW) which is practically always leucine in the4-chain immunoglobulins (98%), the other amino acids at this positionbeing proline (1%) or glutamine (1%).

In the camel heavy-chain immunoglobulin, in the sequences examined atpresent, leucine at position 45 is only found once. It could originatefrom a four-chain immunoglobulin. In the other cases, it is replaced byarginine, cysteine or glutamic acid residue. The presence of chargedamino acids at this position should contribute to making the V_(HH) moresoluble.

The replacement by camelid specific residues such as those of position45 appears to be interesting for the construction of engineered V_(HH)regions derived from the V_(HH) repertoire of 4-chain immunoglobulins.

A second feature specific of the camelid V_(HH) domain is the frequentpresence of a cysteine in the CDR₃ region associated with a cysteine inthe CDR₁ position 31 or 33 or FW₂ region at position 45. The possibilityof establishing a disulphide bond between the CDR₃ region and the restof the variable domain would contribute to the stability and positioningof the binding site.

With the exception-of a single pathogenic myeloma protein (DAW) such adisulphide bond has never been encountered in immunoglobulin V regionsderived from 4 chain immunoglobulins.

The heavy-chain immunoglobulins of the invention have further theparticular advantage of being not sticky. Accordingly theseimmunoglobulins being present in the serum, aggregate much less thanisolated heavy chains of a four-chain immunoglobulins. Theimmunoglobulins of the invention are soluble to a concentration above0.5 mg/ml, preferably above 1 mg/ml and more advantageously above 2mg/ml.

These immunoglobulins further bear an extensive antigen bindingrepertoire and undergo affinity and specificity maturation in vivo.Accordingly they allow the isolation and the preparation of antibodieshaving defined specificity, regarding determined antigens.

Another interesting property of the immunoglobulins of the invention isthat they can be modified and especially humanized. Especially it ispossible to replace all or part of the constant region of theseimmunoglobulins by all or part of a constant region of a human antibody.For example the C_(H) 2 and/or C_(H) 3 domains of the immunoglobulincould be replaced by the C_(H) 2 and/or C_(H) 3 domains of the IgG γ3human immunoglobulin.

In such humanized antibodies it is also possible to replace a part ofthe variable sequence, namely one or more of the framework residueswhich do not intervene in the binding site by human framework residues,or by a part of a human antibody.

Conversely features (especially peptide fragments) of heavy-chainimmunoglobulin V_(HH) regions, could be introduced into the V_(H) orV_(L) regions derived from four-chain immunoglobulins with for instancethe aim of achieving greater solubility of the immunoglobulins.

The invention further relates to a fragment of an immunoglobulin whichhas been described hereabove and especially to a fragment selected fromthe following group:

a fragment corresponding to one heavy polypeptide chain of animmunoglobulin devoid of light chains,

fragments obtained by enzymatic digestion of the immunoglobulins of theinvention, especially those obtained by partial digestion with papainleading to the Fc fragment (constant fragment) and leading to FV_(HH) hfragment (containing the antigen binding sites of the heavy chains) orits dimer F(V_(HH) h)₂, or a fragment obtained by further digestion withpapain of the Fc fragment, leading to the pFc fragment corresponding tothe C-terminal part of the Fc fragment,

homologous fragments obtained with other proteolytic enzymes,

a fragment of at least 10 preferably 20 amino acids of the variableregion of the immunoglobulin, or the complete variable region,especially a fragment corresponding to the isolated V_(HH) domains or tothe V_(HH) dimers linked to the hinge disulphide,

a fragment corresponding to the hinge region of the immunoglobulin, orto at least 6 amino acids of this hinge region,

a fragment of the hinge region comprising a repeated sequence of Pro-X,

a fragment corresponding to at least 10 preferably 20 amino acids of theconstant region or to the complete constant region of theimmunoglobulin.

The invention also relates to a fragment comprising a repeated sequence,Pro-X which repeated sequence contains at least 3 repeats of Pro-X, Xbeing any amino-acid and preferably Gln (glutamine), Lys (lysine) or Glu(acide glutamique); a particular repeated fragment is composed of a12-fold repeat of the sequence Pro-X.

Such a fragment can be advantageously used as a link between differenttypes of molecules.

The amino-acids of the Pro-X sequence are chosen among any natural ornon natural amino-acids.

The fragments can be obtained by enzymatic degradation of theimmunoglobulins. They can also be obtained by expression in cells ororganisms, of nucleotide sequence coding for the immunoglobulins, orthey can be chemically synthesized.

The invention also relates to anti-idiotypes antibodies belonging to theheavy chain immunoglobulin classes. Such anti-idiotypes can be producedagainst human or animal idiotypes. A property of these anti-idiotypes isthat they can be used as idiotypic vaccines, in particular forvaccination against glycoproteins or glycolipids and where thecarbohydrate determines the epitope.

The invention also relates to anti-idiotypes capable of recognizingidiotypes of heavy-chain immunoglobulins.

Such anti-idiotype antibodies can be either syngeneic antibodies orallogenic or xenogeneic antibodies.

The invention also concerns nucleotide sequences coding for all or partof a protein which amino-acid sequence comprises a peptide sequenceselected from the following:

    G G S V Q T G G S L R L S C E I S G L T F D                                                                    SEQ ID NO:1                                     - G G S V Q T G G S L R L S C A V S G F S F S SEQ ID NO:2                     - G G S E Q G G G S L R L S C A I S G Y T Y G SEQ ID NO:3                     - G G S V Q P G G S L T L S C T V S G A T Y S SEQ ID NO:4                     - G G S V Q A G G S L R L S C T G S G F P Y S SEQ ID NO:5                     - G G S V Q A G G S L R L S C V A G F G T S SEQ ID NO:6                       - G G S V Q A G G S L R L S C V S F S P S S SEQ ID NO:7                       - W G Q G T Q V T V S S SEQ ID NO:8                                           - W G Q G T L V T V S S SEQ ID NO:9                                           - W G Q G A Q V T V S S SEQ ID NO:10                                          - W G Q G T Q V T A S S SEQ ID NO:11                                          - R G Q G T Q V T V S L SEQ ID NO:12                                          - A L Q P G G Y C G Y G X - - - - - - - - - - C L SEQ ID NO:62                                                        - V S L M D R I S Q H - - - -                                               - - - - - - - - G C SEQ ID NO:63        - V P A H L G P G A I L D L K K Y - - - - - - K Y SEQ ID NO:64                                                        - F C Y S T A G D G G S G E -                                               - - - - - - - - M Y SEQ ID NO:65        - E L S G G S C E L P L L F - - - - - - - - - D Y SEQ ID NO:66                                                        - D W K Y W T C G A Q T G G Y                                               F - - - - - - - G D SEQ ID NO:67        - R L T E M G A C D A R W A T L A T R T F A Y N Y SEQ ID NO:68                                                        - Q K K D R T R W A E P R E W                                               - - - - - - - - N N SEQ ID NO:69        - G S R F S S P V G S T S R L E S - S D Y - - N Y SEQ ID NO:70                                                        - A D P S I Y Y S I L X I E Y                                               - - - - - - - - K Y SEQ ID NO:71        - D S P C Y M P T M P A P P I R D S F G W - - D D SEQ ID NO:72                                                        - T S S F Y W Y C T T A P Y -                                               - - - - - - - - N V SEQ ID NO:73        - T E I E W Y G C N L R T T F - - - - - - - - T R SEQ ID NO:74                                                        - N Q L A G G W Y L D P N Y W                                               L S V G A Y - - A I SEQ ID NO:75        - R L T E M G A C D A R W A T L A T R T F A Y N Y SEQ ID NO:76                                                        - D G W T R K E G G I G L P W                                               S V Q C E D G Y N Y SEQ ID NO:77        - D S Y P C H L L - - - - - - - - - - - - - - D V SEQ ID NO:78                                                        - V E Y P I A D M C S - - - -                                               - - - - - - - - R Y SEQ ID NO:79        - APELLGGPSVFVFPPKPKDVLSISGXPK SEQ ID NO:39                                   - APELPGGPSVFVFPTKPKDVLSISGRPK SEQ ID NO:40                                   - APELPGGPSVFVFPPKPKDVLSISGRPK SEQ ID NO:41                                   - APELLGGPSVFIFPPKPKDVLSISGRPK SEQ ID NO:42                                   - GQTREPQVYTLAPXRLEL SEQ ID NO:36                                             - GQPREPQVYTLPPSRDEL SEQ ID NO:109                                            - GQPREPQVYTLPPSREEM SEQ ID NO:110                                            - GQPREPQVYTLPPSQEEM SEQ ID NO:111                                            - VTVSSGTNEVCKCPKCPAPELPGGPSVFVFP SEQ ID NO:43                                - or,                                                                         - VTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPAPELLGGPSVFIFP SEQ ID NO:44                                                 - GTNEVCKCPKCP SEQ ID NO:34                                                   - APELPGGPSVFVFP SEQ ID NO:45                                                 - EPKIPQPQPKPQPQPQPQPKPQPKPEPEC                                             TCPKCP SEQ ID NO:38                     - APELLGGPSVFIFP SEQ ID NO:46                                          

Such nucleotide sequences can be deduced from the amino-acid sequencestaking into account the deneneracy of the genetic code. They can besynthesized or isolated from cells producing immunoglobulins of theinvention.

A procedure for the obtention of such DNA sequences is described in theexamples.

The invention also contemplates RNA, especially MRNA sequencescorresponding to these DNA sequences, and also corresponding cDNAsequences.

The nucleotide sequences of the invention can further be used for thepreparation of primers appropriate for the detection in cells orscreening of DNA or cDNA libraries to isolate nucleotide sequencescoding for immunoglobulins of the invention. Such nucleotide sequencescan be used for the preparation of recombinant vectors and theexpression of these sequences contained in the vectors by host cellsespecially prokaryotic cells like bacteria or also eukaryotic cells andfor example CHO cells, insect cells, simian cells like Vero cells, orany other mammalian cells. Especially the fact that the immunoglobulinsof the invention are devoid of light chains permits to secrete them ineukaryotic cells since there is no need to have recourse to the stepconsisting in the formation of the BIP protein which is required in thefour-chain immunoglobulins.

The inadequacies of the known methods for producing monoclonalantibodies or immunoglobulins by recombinant DNA technology comes fromthe necessity in the vast majority of cases to clone simultaneously theV_(H) and V_(L) domains corresponding to the specific binding site of 4chain immunoglobulins. The animals and especially camelids which produceheavy-chain immunoglobulins according to the invention, and possiblyother vertebrate species are capable of producing heavy-chainimmunoglobulins of which the binding site is located exclusively in theV_(HH) domain. Unlike the few heavy-chain immunoglobulins produced inother species by chain separation or by direct cloning, the camelidheavy-chain immunoglobulins have undergone extensive maturation in vivo.Moreover their V region has naturally evolved to function in absence ofthe V_(L). They are therefore ideal for producing monoclonal antibodiesby recombinant DNA technology. As the obtention of specific antigenbinding clones does not depend on a stochastic process necessitating avery large number of recombinant cells, this allows also a much moreextensive examination of the repertoire.

This can be done at the level of the non rearranged V_(HH) repertoireusing DNA derived from an arbitrarily chosen tissue or cell type or atthe level of the rearranged V_(HH) repertoire, using DNA obtained from Blymphocytes. More interesting however is to transcribe the mRNA fromantibody producing cells and to clone the cDNA with or without prioramplification into an adequate vector. This will result in the obtentionof antibodies which have already undergone affinity maturation.

The examination of a large repertoire should prove to be particularlyuseful in the search for antibodies with catalytic activities.

The invention thus provides libraries which can be generated in a waywhich includes part of the hinge sequence, the identification is simpleas the hinge is directly attached to the V_(HH) domain.

These libraries can be obtained by cloning cDNA from lymphoid cells withor without prior PCR amplification. The PCR primers are located in thepromoter, leader or framework sequences of the V_(HH) for the 5' primerand in the hinge, CH₂, CH₃, 3' untranslated region or polyA tail for the3' primer. A size selection of amplified material allows theconstruction of a library limited to heavy chain immunoglobulins.

In a particular example, the following 3' primer in which a KpnI sitehas been constructed and which corresponds to amino-acids 313 to 319(CGC CAT CAA GGT AAC AGT TGA) SEQ ID NO: 47 is used in conjunction withmouse V_(HH) primers described by Sestry et al and containing a Xho site

    AG GTC CAG CTG CTC GAG TCT GG                                                                        SEQ ID NO:48                                              - AG CTC CAG CTG CTC GAG TCT GG SEQ ID NO:49                                  - AG GTC CAG CTT CTC GAG TCT GG SEQ ID NO:50                                  -                XhoI site                                             

These primers yield a library of camelid heavy chain immunoglobulinscomprising the V_(HH) region (related to mouse or human subgroup III),the hinge and a section of CH₂.

In another example, the cDNA is polyadenylated at its 5' end and themouse specific V_(HH) primers are replaced by a poly T primer with aninbuilt XhoI site, at the level of nucleotide 12.

CTCGAGT₁₂.

The same 3' primer with a KpnI site is used.

This method generates a library containing all subgroups ofimmunoglobulins.

Part of the interest in cloning a region encompassing the hinge-CH₂ linkis that in both γ2 and γ3, a Sac site is present immediately after thehinge. This site allows the grafting of the sequence coding for theV_(HH) and the hinge onto the Fc region of other immunoglobulins, inparticular the human IgG₁ and IgG₃ which have the same amino acidsequence at this site (Glu₂₄₆ LeU₂₄₇).

As an example, the invention contemplates a cDNA library composed ofnucleotide sequences coding for a heavy-chain immunoglobulin , such asobtained by performing the following steps:

a) treating a sample containing lymphoid cells, especially periferal,lymphocytes, spleen cells, lymph nodes or another lyphoid tissue from ahealthy animal, especially selected among the Camelids, in order toseparate the lymphoid cells,

b) separating polyadenylated RNA from the other nucleic acids andcomponents of the cells,

c) reacting the obtained RNA with a reverse transcriptase in order toobtain the corresponding cDNA,

d) contacting the cDNA of step c) with 5' primers corresponding to mouseV_(H) domain of four-chain immunoglobulins, which primer contains adetermined restriction site, for example an XhoI site and with 3'primers corresponding to the N-terminal part of a C_(H) 2 domaincontaining a KpnI site,

e) amplifying the DNA,

f) cloning the amplified sequence in a vector, especially in abluescript vector,

g) recovering the clones hybridizing with a probe corresponding to thesequence coding for a constant domain from an isolated heavy-chainimmunoglobulin.

This cloning gives rise to clones containing DNA sequences including thesequence coding for the hinge. It thus permits the characterization ofthe subclass of the immunoglobulin and the SacI site useful for graftingthe FV_(HH) h to the Fc region.

The recovery of the sequences coding for the heavy-chain immunoglobulinscan also be achieved by the selection of clones containing DNA sequenceshaving a size compatible with the lack of the C_(H) 1 domain.

It is possible according to another embodiment of the invention, to addthe following steps between steps c) and d) of the above process:

in the presence of a DNA polymerase and of deoxyribonucleotidetriphosphates, contacting said cDNA with oligonucleotide degeneratedprimers, which sequences are capable of coding for the hinge region andN-terminal V_(HH) domain of an immunoglobulin, the primers being capableof hybridizing with the cDNA and capable of initiating the extension ofa DNA sequence complementary to the cDNA used as template,

recovering the amplified DNA.

The clones can be expressed in several types of expression vectors. Asan example using a commercially available vector Immuno PBS (Huse et al:Science (1989) 246, 1275), clones produced in Bluescript® according tothe above described procedure, are recovered by PCR using the same XhoIcontaining 5' primer and a new 3' primer, corresponding to residues113-103 in the framework of the immunoglobulins, in which an Spe sitehas been constructed: TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO:51. This procedure allows the cloning of the V_(HH) in the Xho/Spe siteof the Immuno PBS vector. However, the 3' end of the gene is not inphase with the identification "tag" and the stop codon of the vector. Toachieve this, the construct is cut with Spe and the 4 base overhangs arefilled in, using the Klenow fragment after which the vector isreligated. A further refinement consists in replacing the marker ("tag")with a poly histidine so that metal purification of the cloned V_(HH)can be performed. To achieve this a Spe/EcoRI double strandedoligo-nucleotide coding for 6 histidines and a termination codon isfirst constructed by synthesis of both strands followed by heating andannealing:

    CTA GTG CAC CAC CAT CAC CAT CAC TAA* TAG* SEQ ID NO: 52

    AC GTG GTG GTA GTG GTA GTG ATT ATC TTA A SEQ ID NO: 53

The vector containing the insert is then digested with SpeI and EcoRI toremove the resident "tag" sequence which can be replaced by thepoly-His/termination sequence. The produced V_(HH) can equally bedetected by using antibodies raised against the dromedary V_(HH)regions. Under laboratory conditions, V_(HH) regions are produced in theImmuno PBS vector in mg amounts per liter.

The invention also relates to a DNA library composed of nucleotidesequences coding for a heavy-chain immunoglobulin, such as obtained fromcells with rearranged immunoglobulin genes.

In a preferred embodiment of the invention, the library is prepared fromcells from an animal previously immunized against a determined antigen.This allows the selection of antibodies having a preselected specificityfor the antigen used for immunization.

In another embodiment of the invention, the amplification of the cDNA isnot performed prior to the cloning of the cDNA.

The heavy-chain of the four-chain immunoglobulins remains sequestered inthe cell by a chaperon protein (BIP) until it has combined with a lightchain. The binding site for the chaperon protein is the C_(H) 1 domain.As this domain is absent from the heavy chain immunoglobulins, theirsecretion is independent of the presence of the BIP protein or of thelight chain. Moreover the inventors have shown that the obtainedimmunoglobulins are not sticky and accordingly will not abnormallyaggregate.

The invention also relates to a process for the preparation of amonoclonal antibody directed against a determined antigen, the antigenbinding site of the antibody consisting of heavy polypeptide chains andwhich antibody is further devoid of light polypeptide chains, whichprocess comprises:

immortalizing lymphocytes, obtained for example from the peripheralblood of Camelids previously immunized with a determined antigen, withan immortal cell and preferably with myeloma cells, in order to form ahybridoma,

culturing the immortalized cells (hybridoma) formed and recovering thecells producing the antibodies having the desired specificity.

The preparation of antibodies can also be performed without a previousimmunization of Camelids.

According to another process for the preparation of antibodies, therecourse to the technique of the hybridoma cell is not required.

According to such process, antibodies are prepared in vitro and they canbe obtained by a process comprising the steps of:

cloning into vectors, especially into phages and more particularlyfilamentous bacteriophages, DNA or cDNA sequences obtained fromlymphocytes especially PBLs of Camelids previously immunized withdetermined antigens,

transforming prokaryotic cells with the above vectors in conditionsallowing the production of the antibodies,

selecting the antibodies for their heavy-chain structure and further bysubjecting them to antigen-affinity selection,

recovering the antibodies having the desired specificity,

In another embodiment of the invention the cloning is performed invectors, especially into plasmids coding for bacterial membraneproteins. Procaryotic cells are then transformed with the above vectorsin conditions allowing the expression of antibodies in their membrane.

The positive cells are further selected by antigen affinity selection.

The heavy chain antibodies which do not contain the C_(H) 1 domainpresent a distinct advantage in this respect. Indeed, the C_(H) 1 domainbinds to BIP type chaperone proteins present within eukaryotic vectorsand the heavy chains are not transported out of the endocytoplasmicreticulum unless light chains are present. This means that in eukaryoticcells, efficient cloning of 4-chain immunoglobulins in non mammaliancells such as yeast-cells can depend on the properties of the residentBIP type chaperone and can hence be very difficult to achieve. In thisrespect the heavy chain antibodies of the invention which lack the CH₁domain present a distinctive advantage.

In a preferred embodiment of the invention the cloning can be performedin yeast either for the production of antibodies or for the modificationof the metabolism of the yeast. As example, Yep 52 vector can be used.This vector has the origin of replication (ORI) 2μ of the yeast togetherwith a selection marker Leu 2.

The cloned gene is under the control of gall promoter and accordingly isinducible by galactose. Moreover, the expression can be repressed byglucose which allows the obtention of very high concentration of cellsbefore the induction.

The cloning between BamHI and SalI sites using the same strategy ofproduction of genes by PCR as the one described above, allows thecloning of camelid immunoglobulin genes in E. coli. As example ofmetabolic modulation which can be obtained by antibodies and prcopsedfor the yeast, one can site the cloning of antibodies directed againstcyclins, that is proteins involved in the regulation of the cellularcycle of the yeast (TIBS 16 430 J. D. Mc Kinney, N. Heintz 1991).Another example is the introduction by genetic engineering of anantibody directed against CD₂₈, which antibody would be inducible (forinstance by gall), within the genome of the yeast. The CD₂₈ is involvedat the level of the initiation of cell division, and therefore theexpression of antibodies against this molecule would allow an efficientcontrol of multiplication of the cells and the optimization of methodsfor the production in bioreactors or by means of immobilized cells.

In yet another embodiment of the invention, the cloning vector is aplasmid or a eukaryotic virus vector and the cells to be transformed areeukaryotic cells, especially yeast cells, mammalian cells for exampleCHO cells or simian cells such as Vero cells, insect cells, plant cells,or protozoan cells.

For more details concerning the procedure to be applied in such a case,reference is made to the publication of Marks et al, J. Mol. Biol. 1991,222:581-597.

Furthermore, starting from the immunoglobulins of the invention, or fromfragments thereof, new immunoglobulins or derivatives can be prepared.

Accordingly immunoglobulins replying to the above given definitions canbe prepared against determined antigens. Especially the inventionprovides monoclonal or polyclonal antibodies devoid of light polypeptidechains or antisera containing such antibodies and directed againstdetermined antigens and for example against antigens of pathologicalagents such as bacteria, viruses or parasites. As example of antigens orantigenic determinants against which antibodies could he prepared, orecan cite the envelope glycoproteins of viruses or peptides thereof, suchas the external envelope glycoprotein of a HIV virus, the surfaceantigen of the hepatitis B virus.

Immunoglobulins of the invention can also be directed against a protein,hapten, carbohydrate or nucleic acid.

Particular antibodies according to the invention are directed againstthe galactosylα-1-3-galactose epitope.

The immunoglobulins of the invention allow further the preparation ofcombined products such as the combination of the heavy-chainimmunoglobulin or a fragment thereof with a toxin, an enzyme, a drug, ahormone.

As example one can prepare the combination of a heavy-chainimmunoglobulin bearing an antigen binding site recognizing a myelomaimmunoglobulin epitope with the abrin or mistletoe lectin toxin. Such aconstruct would have its uses in patient specific therapy.

Another advantageous combination is that one can prepare between aheavy-chain immunoglobulins recognizing an insect gut antigen with atoxin specific for insects such as the toxins of the different serotypesof Bacillus thuringiensis or Bacillus sphaericus. Such a constructcloned into plants can be used to increase the specificity or the hostrange of existing bacterial toxins.

The invention also proposes antibodies having different specificities oneach heavy polypeptide chains. These multifunctional, especiallybifunctional antibodies could be prepared by combining two heavy chainsof immunoglobulins of the invention or one heavy chain of animmunoglobulin of the invention with a fragment of a four-chain modelimmunoglobulin.

The invention also provides hetero-specific antibodies which can be usedfor the targetting of drugs or any biological substance like hormones.In particular they can be used to selectively target hormones orcytokines to a limited category of cells. Examples are a combination ofa murine or human antibody raised against interleukin 2 (IL₂) and aheavy-chain antibody raised against CD₄ cells. This could be used toreactivate CD₄ cells which have lost their IL₂ receptor.

The heavy-chain immunoglobulins of the invention can also be used forthe preparation of hetero-specific antibodies. These can be achievedeither according to the above described method by reduction of thebridges between the different chains and reoxydation, according to theusual techniques, of two antibodies having different specificities, butit can also be achieved by serial cloning of two antibodies for instancein the Immuno pBS vector.

In such a case, a first gene corresponding to the V_(HH) domaincomprised between Xho site and a Spe site is prepared as describedabove. A second gene is then prepared through an analogous way by usingas 5' extremity a primer containing a Spe site, and as 3' extremity aprimer containing a termination codon and an EcoRI site. The vector isthen digested with EcoRI and XhoI and further both V_(HH) genes aredigested respectively by Xho/Spe and by Spe/EcoRI.

After ligation, both immunoglobulin genes are serially cloned. Thespacing between both genes can be increased by the introduction ofaddition codons within the 5' SpeI primer.

In a particular embodiment of the invention, the hinge region of IgG2immunoglobulins according to the invention is semi-rigid and is thusappropriate for coupling proteins. In such an application proteins orpeptides can be linked to various substances, especially to ligandsthrough the hinge region used as spacer. Advantageously the fragmentcomprises at least 6 amino acids.

According to the invention it is interesting to use a sequencecomprising a repeated sequence Pro-X, X being any amino-acid andpreferably Gln, Lys or Glu, especially a fragment composed of at least a3-fold repeat and preferably of a 12-fold repeat, for coupling proteinsto ligand, or for assembling different protein domains.

The hinge region or a fragment thereof can also be used for couplingproteins to ligands or for assembling different protein domains.

Usual techniques for the coupling are appropriate and especiallyreference may be made to the technique of protein engineering byassembling cloned sequences.

The antibodies according to this invention could be used as reagents forthe diagnosis in vitro or by imaging techniques. The immunoglobulins ofthe invention could be labelled with radio-isotopes, chemical orenzymatic markers or chemiluminescent markers.

As example and especially in the case of detection or observation withthe immunoglobulins by imaging techniques, a label like technetium,especially technitium 99 is advantageous. This label can be used fordirect labelling by a coupling procedure with the immunoglobulins orfragments thereof or for indirect labelling after a step of preparationof a complex with the technitium.

Other interesting radioactive labels are for instance indium andespecially indium 111, or iodine, especially I¹³¹, I¹²⁵ and I¹²³.

For the description of these techniques reference is made to the FRpatent application published under number 2649488.

In these applications the small size of the V_(HH) fragment is adefinitive advantage for penetration into tissue.

The invention also concerns monoclonal antibodies reacting withanti-idiotypes of the above-described antibodies.

The invention also concerns cells or organisms in which heavy-chainimmunoglobulins have been cloned. Such cells or organisms can be usedfor the purpose of producing heavy-chain immunoglobulins having adesired preselected specificity, or corresponding to a particularrepertoire. They can also be produced for the purpose of modifying themetabolism of the cell which expresses them. In the case of modificationof the metabolism of cells transformed with the sequences coding forheavy-chain immunoglobulins, these produced heavy-chain immunoglobulinsare used like antisense DNA. Antisense DNA is usually involved inblocking the expression of certain genes such as for instance thevariable surface antigen of trypanosomes or other pathogens. Likewise,the production or the activity of certain proteins or enzymes could beinhibited by expressing antibodies against this protein or enzyme withinthe same cell.

The invention also relates to a modified 4-chain immunoglobulin orfragments thereof, the V_(H) regions of which has been partiallyreplaced by specific sequences or amino acids of heavy chainimmunoglobulins, especially by sequences of the V_(HH) domain. Aparticular modified V_(H) domain of a four-chain immunoglobulin, ischaracterized in that the leucine, proline or glutamine in position 45of the V_(H) regions has been replaced by other amino acids andpreferably by arginine, glutamic acid or cysteine.

A further modified V_(H) or V_(L) domain of a four-chain immunoglobulin,is characterized by linking of CDR loops together or to FW regions bythe introduction of paired cysteines, the CDR region being selectedbetween the CDR₁ and the CDR₃, the FW region being the FW₂ region, andespecially in which one of the cysteines introduced is in position 31,33 of the CDR₁ or 45 of FW₂.

Especially the introduction of paired cysteines is such that the CDR₃loop is linked to the FW2 or CDR1 domain and more especially thecysteine of the CDR3 of the V_(H) is linked to a cysteine in position31, 33 of the CDR₁ or in position 45 of FW₂.

In another embodiment of the invention, plant cells can be modified bythe heavy-chain immunoglobulins according to the invention, in orderthat they acquire new properties or increased properties.

The heavy-chain immunoglobulins of the invention can be used for genetherapy of cancer for instance by using antibodies directed againstproteins present on the tumor cells.

In such a case, the expression of one or two V_(HH) genes can beobtained by using vectors derived from parvo or adeno viruses. The parvoviruses are characterized by the fact that they are devoid ofpathogenicity or almost not pathogenic for normal human cells and by thefact that they are capable of easily multiplying in cancer cells (RusselS. J. 1990, Immunol. Today II. 196-200).

The heavy-chain immunoglobulins are for instance cloned withinHindIII/XbaI sites of the infectious plasmid of the murine MVM virus(pMM984). (Merchlinsky et al, 1983, J. Virol. 47, 227-232) and thenplaced under the control of the MVM38 promoter.

The gene of the V_(HH) domain is amplified by PCR by using a 5' primercontaining an initiation codon and a HindIII site, the 3' primercontaining a termination codon and a XbaI site.

This construct is then inserted between positions 2650 (HindIII) and4067 (XbaI) within the plasmid.

The efficiency of the cloning can be checked by transfection. The vectorcontaining the antibody is then introduced in permissive cells (NB-E) bytransfection.

The cells are recovered after two days and the presence of V_(HH)regions is determined with an ELISA assay by using rabbit antiserumreacting with the V_(HH) part.

The invention further allows the preparation of catalytic antibodiesthrough different ways. The production of antibodies directed againstcomponents mimicking activated states of substrates (as example vanadateas component mimicking the activated state of phosphate in order toproduce their phosphoesterase activities, phosphonate as compoundmimicking the peptidic binding in order to produce proteases) permits toobtain antibodies having a catalytic function. Another way to obtainsuch antibodies consists in performing a random mutagenesis in clones ofantibodies for example by PCR, in introducing abnormal bases during theamplification of clones. These amplified fragments obtained by PCR arethen introduced within an appropriate vector for cloning. Theirexpression at the surface of the bacteria permits the detection by thesubstrate of clones having the enzymatic activity. These two approachescan of course be combined. Finally, on the basis of the data availableon the structure, for example the data obtained by XRay crystallographyor NMR, the modifications can be directed. These modifications can beperformed by usual techniques of genetic engineering or by completesynthesis. One advantage of the V_(HH) of the heavy chainimmunoglobulins of the invention is the fact that they are sufficientlysoluble.

The heavy chain immunoglobulins of the invention can further be producedin plant cells, especially in transgenics plants. As example the heavychain immunoglobulins can be produced in plants using the pMon530plasmid (Roger et al. Meth Enzym 153 1566 1987) constitutive plantexpression vector as has been described for classical four chainantibodies (Hiat et al. Nature 342 76-78, 1989) once again using theappropriate PCR primers as described above, to generate a DNA fragmentin the right phase.

Other advantages and characteristics of the invention will becomeapparent in the examples and figures which follow.

FIGURES

FIGS. 1(A), (B), and (C): Characterisation and purification of camel IgGby affinity chromatography on Protein A and Protein G sepharose(Pharmacia)

FIG. 1(A) shows, after reduction, the SDS-PAGE protein profile of theadsorbed and non adsorbed fractions of Camelus dromedarius serum. Thefraction adsorbed on Protein A and eluted with NaCl 0.15 M acetic acid0.58% show upon reduction (lane c) three heavy chain components ofrespectively 50, 46 and 43 Kd and light chain (rabbit IgG in lane a).The fractions adsorbed on a Protein G Sepharose (Pharmacia) derivativewhich has been engineered to delete the albumin binding region (lane e)and eluted with 0.1 M gly HCl pH 2.7 lacks the 46 Kd heavy chain whichis recovered in the non adsorbed fraction (lane f). None of thesecomponents are present in the fraction non adsorbed on Protein A (laned), lane b contains the molecular weight markers.

FIGS. 1(B) and (C) By differential elution, immunoglobulin fractionscontaining the 50 and 43 Kd heavy chain can be separated. 5 ml of C.dromadarius serum is adsorbed onto a 5 ml Protein G sepharose column andthe column is extensively washed with 20 mM phosphate buffer, pH 7.0.Upon elution with pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid) a 100Kd component is eluted which upon reduction yields a 43 Kd heavy chain,(lane 1). After column eluant absorbance has fallen to background levela second immunoglobulin component of 170 Kd can be eluted with pH 2.7buffer (0.1 M glycine HC). This fraction upon reduction yields a 50 Kdheavy chain and a board light chain band (lane 2). The fraction nonadsorbed on Protein G is then brought on a 5 ml Protein A Sepharosecolumn. After washing and elution with pH 3.5 buffer (0.15 M NaCl, 0.58%acetic acid) a third immunoglobulin of 100 Kd is obtained which consistssolely of 46 Kd heavy chains (lane 3).

FIGS. 2(A) and (B): Immunoglobulins of Camelus bactrianus, Lama vicugna,Lama glama and Lama pacos to Protein A (A lanes) and to Protein G (Glanes) analyzed on SDS-PAGE before FIG. 2(A) and after reduction FIG.2(B).

10 μl of serum obtained from the different species were added toEppendorf^(R) tubes containing 10 mg of Protein A or Protein G sepharosesuspended in 400 μl of pH 8.3 immunoprecipitation buffer (NaCl 0.2. M,Tris 0.01 M; EDTA 0.01 M, Triton X100 1%, ovalbumin 0.1%). The tubeswere slowly rotated for 2 hours at 4° C. After centrifugation thepellets were washed 3 times in buffer and once in buffer in which theTriton and ovalbumin had been ommitted. The pellets were thenresuspended in the SDS-PAGE sample solution 70 μl per pellet with orwithout dithiotreitol as reductant. After boiling for 3 min at 100° C.,the tubes were centrifuged and the supernatants analysed.

In all species examined the unreduced fractions FIG. 2(A) contain inaddition to molecules of approximately 170 Kd also smaller majorcomponents of approximately 100 Kd. In the reduced sample FIG. 2(B) theconstituant heavy and light chains are detected. In all species a heavychain component (marked by an asterisk *) is present in the materialeluted from the Protein A but absent in the material eluted from theProtein G.

FIGS. 3(A) and (B): IgG₁, IgG₂ and IgG₃ were prepared from serumobtained from healthy or Trypanosama evansi infected Camelus dromedarius(CATT titer 1/160 (3) and analysed by radioimmunopreci- pitation orWestern Blotting for anti trypanosome activity

FIG. 3(A) ³⁵ S methionine labelled Trypanosome evansi antigens lysate(500.000 counts) was added to Eppendorf tubes containing 10 μl of serumor, 20 μg of IgG₁, IgG₂ or IgG₃ in 200 μl of pH 8.3 immunoprecipitationbuffer containing 0.1 M TLCK as proteinase inhibitor and slowly rotatedat 4° C. during one hour. The tubes were then supplemented with 10 mg ofProtein A Sepharose suspended in 200 μl of the same pH 8.3 buffer andincubated at 4° C. for an additional hour.

After washing and centrifugation at 15000 rpm for 12 s, each pellet wasresuspended in 75 μl SDS-PAGE sample solution containing DTT and heatedfor 3 min. at 100° C. After centrifugation in an Eppendorf minifuge at15000 rpm for 30 s, 5 μl of the supernatant was saved for radioactivitydetermination and the reminder analysed by SDS-PAGE and fluorography.The counts/5 μl sample are inscribed on for each line.

FIG. 3(B) 20 μg of IgG₁, IgG₂ and IgG₃ from healthy and trypanosomeinfected animals were separated by SDS-PAGE without prior reduction orheating. The separated samples were then electro transferred to anitrocellulose membrane, one part of the membrane was stained withPonceau Red to localise the protein material and the reminder incubatedwith 1% ovalbumin in TST buffer (Tris 10 mM, NaCl 150 mM, Tween 0.05%)to block protein binding sites.

After blocking, the membrane was extensively washed with TST buffer andincubated for 2 hours with ³⁵ S-labelled trypanosome antigen. Afterextensive washing, the membrane was dried and analysed byautoradiography. To avoid background and unspecific binding, thelabelled trypanosome lysate was filtered through a 45μ millipore filterand incubated with healthy camel immunoglobulin and ovalbumin adsorbedon a nitrocellulose membrane.

FIGS. 4(A) and (B): Purified IgG3 of the camel, by affinitychromatography on Protein A Sepharose are partially digested with papainand separated on Protein A sepharose.

14 mg of purified IgG3 were dissolved in 0.1M phosphate buffer pH 7.0containing 2 mM EDTA. They were digested by 1 hour incubation at 37° C.with mercurypapain (1% enzyme to protein ratio) activated by 5.10⁴ Mcysteine. The digestion was blocked by the addition of excessiodoacetamide (4.10² M) (13). After centrifugation of the digest in anependorf centrifuge for 5 min at 15000 rpm, the papain fragments wereseparated on a protein A Sepharose column into binding (B) and nonbinding (NB) fractions. The binding fraction was eluted from the columnwith 0.1M glycine HCl buffer pH 1.7.

FIG. 5: Schematic presentation of a model for IgG3 molecules devoid oflight chains.

FIG. 6: Schematic representation of immuno-globulins having heavypolypeptide chains and devoid of light chains, regarding conventionalfour-chain model immunoglobulin

Representation of a hinge region.

FIG. 7: Alignment of 17 V_(HH) DNA sequences of Camel heavy chainimmunoglobulins SEQ ID NOS: 92-108

FIG. 8: Expression and purification of the camel V_(HH) 21 protein fromE. coli

I HEAVY CHAIN ANTIBODIES IN CAMELIDS

When Camelus dromedarius serum is adsorbed on Protein G sepharose, anappreciable amount (25-35%) of immunoglobulins (Ig) remains in solutionwhich can then be recovered by affinity chromatography on Protein Asepharose (FIG. 1A). The fraction adsorbed on Protein G can bedifferentially eluted into a tightly bound fraction (25%) consisting ofmolecules of an unreduced apparent molecular weight (MW) of 170 Kd and amore weakly bound fraction (30-45%) having an apparent molecular weightof 100 Kd (FIG. 1B). The 170 Kd component when reduced yields 50 Kdheavy chains and large 30 Kd light chains. The 100 Kd fraction istotally devoid of light chains and appears to be solely composed ofheavy chains which after reduction have on apparent MW of 43 Kd (FIG.1C). The fraction which does not bind to Protein G can be affinitypurified and eluted from a Protein A column as a second 100 Kd componentwhich after reduction appears to be composed solely of 46 Kd heavychains.

The heavy chain immoglobulins devoid of light chains total up to 75% ofthe molecules binding to Protein A.

As all three immunoglobulins bind to Protein A we refer to them as IgG:namely IgG, (light chain and heavy chain γ1 (50 Kd) binding to ProteinG, IgG₂ (heavy chain γ2 (46 Kd) non binding to Protein G and IgG₃ (heavychain γ3 (43 Kd) binding to Protein G. There is a possibility that thesethree sub(classes) can be further subdivided.

A comparative study of old world camelids (Camelus bactrianus andCamelus dromedarius) and new world camelids (Lama pacos, Lama glama,Lama vicugna) showed that heavy chain immunoglobulins are found in allspecies examined, albeit with minor differences in apparent molecularweight and proportion. The new world camelids differs from the old worldcamelids in having a larger IgG₃ molecule (heavy chain immunoglobulinbinding to Protein G) in which the constituant heavy chains have anapparent molecular weight of 47 Kd (FIGS. 2A and B).

The abundance of the heavy chain immunoglobulins in the serum ofcamelids raises the question of what their role is in the immuneresponse and in particular whether they bear antigen binding specificityand if so how extensive is the repertoire. This question could beanswered by examining the immunoglobulins from Trypanosoma evansiinfected camels (Camelus dromedarius).

For this purpose, the corresponding fractions of IgG₁, IgG₂, IgG₃ wereprepared from the serum of a healthy camel and from the serum of camelswith a high antitrypanosome titer, measured by the Card AgglutinationTest (3). In radio-immunoprecipitation, IgG₁, IgG₂ and IgG₃ derived frominfected camel indicating extensive repertoire heterogeneity andcomplexity (FIG. 3A) were shown to bind a large number of antigenspresent in a ³⁵ S methionine labelled trypanosome lysate.

In blotting experiments ³⁵ S methionine labelled trypanosome lysatebinds to SDS PAGE separated IgG₁, IgG₂ and IgG₃ obtained from infectedanimals (FIG. 3B).

This leads us to conclude that the camelid heavy chain IgG₂ and IgG₃ arebona fide antigen binding antibodies.

An immunological paradigm states that an extensive antibody repertoireis generated by the combination of the light and heavy chain variable Vregion repertoires (6). The heavy chain immunoglobulins of the camelseem to contradict this paradigm.

Immunoglobulins are characterized by a complex I.E.F. (isoelectricfocussing) pattern reflecting their extreme heterogeneity. To determinewhether the two heavy chains constituting the IgG₂ and IgG₃ areidentical or not, the isoelectric focussing (I.E.F.) pattern wereobserved before and after chain separation by reduction and alkylationusing iodoacetamide as alkylating agent.

As this alkylating agent does not introduce additional charges in themolecule, the monomers resulting from the reduction and alkylation of aheavy chain homodimer will have practically the same isolectric point asthe dimer, whereas if they are derived from a heavy chain heterodimer,the monomers will in most cases differ sufficiently in isoelectric pointto generate a different pattern in I.E.F.

Upon reduction, and alkylation by iodoacetamide the observed pattern isnot modified for the Camelus dromedarius IgG₂ and IgG₃ indicating thatthese molecules are each composed of two identical heavy chains whichmigrate to the same position as the unreduced molecule they originatedfrom.

In contrast, the I.E.F. pattern of IgG₁ is completely modified afterreduction as the isoelectric point of each molecule is determined by thecombination of the isoelectric points of the light and heavy chainswhich after separation will each migrate to a different position.

These findings indicate that the heavy chains alone can generate anextensive repertoire and question the contribution of the light chain tothe useful antibody repertoire. If this necessity be negated, what otherrole does the light chain play.

Normally, isolated heavy chain from mammalian immunoglobulins tend toaggregate considerably but are only solubilized by light chains (8, 9)which bind to the C_(H) 1 domain of the heavy chain.

In humans and in mice a number of spontaneous or induced myelomasproduce a pathological immunoglobulin solely composed of heavy chains(heavy chain disease). These myeloma protein heavy chains carrydeletions in the C_(H) 1 and V_(HH) domains (10). The reason why fulllength heavy chains do not give rise to secreted heavy chain in suchpathological immunoglobulins seems to stem from the fact that thesynthesis of Ig involves a chaperoning protein, the immunoglobulin heavychain binding protein or BIP (11), which normally is replaced by thelight chain (12). It is possible that the primordial role of the lightchain in the four-chain model immunoglobulins is that of a committedheavy chain chaperon and that the emergence of light chain repertoireshas just been an evolutionary bonus.

The camelid γ2 and γ3 chains are considerably shorter than the normalmammalian γ chain. This would suggest that deletions have occurred inthe C_(H) 1 domain. Differences in sizes of the γ2 and γ3immunoglobulins of old and new world camelids suggests that deletionsoccurred in several evolutionary steps especially in the C_(H) 1 domain.

II The Heavy Chain Immunoglobulins of the Camelids Lack The C_(H) 1Domain

The strategy followed for investigating the heavy chain immunoglobulinprimary structure is a combination of protein and cDNA sequencing; theprotein sequencing is necessary to identify sequence stretchescharacteristic of each immunoglobulin. The N-terminal of theimmunoglobulin being derived from the heavy chain variable regionrepertoire only yields information on the V_(HH) subgroups (variableregion of the heavy chain) and cannot be used for class or subclassidentification. This means that sequence data had to be obtained frominternal enzymatic or chemical cleavage sites.

A combination of papain digestion and Protein A affinity chromatographyallowed the separation of various fragments yielding information on thegeneral structure of IgG3.

The IgG3 of the camel (Camelus dromedarius) purified by affinitychromatography on Protein A Sepharose were partially digested withpapain and the digest was separated on Protein A Sepharose into bindingand non binding fractions. These fractions were analysed by SDS PAGEunder reducing and non reducing conditions (FIGS. 4A and B).

The bound fraction contained two components, one of 28 Kd and one of14.4 Kd, in addition to uncleaved or partially cleaved material. Theywere well separated by gel electrophoresis (from preparative 19%SDS-PAGE gels) under non reducing conditions and were further purifiedby electroelution (in 50 nM amonium bicarbonate, 0.1% (w/v) SDS using aBioRad electro-eluter). After lyophilization of these electroelutedfractions, the remaining SDS was eliminated by precipitating the proteinby the addition of 90% ethanol, mixing and incubating the mixtureovernight at -20° C. (14). The precipitated protein was collected in apellet by centrifuging (15000 rpm, 5 min) and was used for proteinsequencing. N-terminal sequencing was performed using the automatedEdman chemistry of an Applied Biosystem 477A pulsed liquid proteinsequencer. Amino acids were identified as their phenylthiohydantoin(PTH) derivatives using an Applied Biosystem 120 PTH analyser. Allchemical and reagents were purchased from Applied Biosystems. Analysisof the chromatographic data was performed using Applied Biosystemssoftware version 1.61. In every case the computer aided sequenceanalysis was confirmed by direct inspection of the chronatograns fromthe PTH analyser. Samples for protein sequencing were dissolved ineither 50% (v/v) trifluoroacetic acid(TFA) (28 Kd fragment) or 100% TFA(14 Kd fragment). Samples of dissolved protein equivalent to 2000 pmol(28 Kd fragment) or 500 pmol (14 Kd fragment) were applied toTFA-treated glass fibre discs. The glass fibre discs were coated withBioBrene (3 mg) and precycled once before use.

N-terminal sequencing of the 28 Kd fragment yields a sequence homologousto the N-terminal part of γ C_(H) 2 domain and hence to the N-terminalend of the Fc fragment. The N-terminal sequence of the 14.4 Kd fragmentcorresponds to the last lysine of a γ C_(H) 2 and the N-terminal end ofa γ C_(H) 3 domain (Table 1). The molecular weight (MW) of the papainfragments and the identification of their N-terminal sequences led us toconclude that the C_(H) 2 and C_(H) 3 domains of the γ3 heavy chains arenormal in size and that the deletion must occur either in the C_(H) 1 orin the V_(HH) domain to generate the shorted γ3 chain. The fractionswhich do not bind to Protein A Sepharose contain two bands of 34 and 17Kd which are more diffuse is SDS PAGE indicating that they originatefrom the variable N-terminal part of the molecule (FIGS. 4A and B).

Upon reduction, a single diffuse band of 17 Kd is found indicating thatthe 34 Kd is a disulfide bonded dimer of the 17 Kd component. The 34 Kdfragment apparently contains the hinge and the N-terminal domain V_(HH).

The protein sequence data can be used to construct degenerateoligonucleotide primers allowing PCR amplification of cDNA or genomicDNA.

It has been shown that the cells from camel spleen imprint cells reactedwith rabbit and anti camel immunoglobulin sera and that the spleen washence a site of synthesis of at least one immunoglobulin class. cDNA wastherefore synthetised from camel spleen mRNA. The conditions for theisolation of RNA were the following: total RNA was isolated from thedromedary spleen by the guanidium isothiocyanate method (15). mRNA waspurified with oligo T-paramagnetic beads. cDNA synthesis is obtainedusing 1 pg mRNA template, an oligodT primer and reverse transcriptase(BOERHINGER MAN). Second strand cDNA is obtained using RNAse H and E.coli DNA polymerase I according to the condition given by the supplier.

Relevant sequences were amplified by PCR: 5 ng of cDNA was amplified byPCR in a 100 μl reaction mixture (10 mM Tris-HCl pH 8.3, 50 mM KCl, 15mM MgCl₂, 0.01% (w/v) gelatine, 200 μM of each dNTP and 25 pmoles ofeach primer) overlaid with mineral oil (Sigma).

Degenerate primers containing EcoRI and KpnI sites and further clonedinto pUC 18. After a round of denaturing and annealing (94° C. for 5 minand 54° C. for 5 min), 2 units of Taq DNA polymerase were added to thereaction mixture before subjecting it to 35 cycles of amplification: 1min at 94° C. (denature) 1 min at 54° C. (anneal), 2 min at 72° C.(elongate). To amplify DNA sequences between V_(HH) and C_(H) 2 domains, (# 72 clones), the PCR was performed in the same conditions with theexception that the annealing temperature was increased to 60° C.

One clone examined (#56/36) had a sequence corresponding to theN-terminal part of a C_(H) 2 domain identical to the sequence of the 28Kd fragment. The availability of this sequence data allowed theconstruction of an exact 3' primer and the cloning of the region betweenthe N-terminal end of the V_(HH) and the C_(H) 2 domain.

5' primers corresponding to the mouse V_(HH) (16) and containing a XhoIrestriction site were used in conjunction with the 3' primer in which aKpnI site had been inserted and the amplified sequences were cloned intopbluescript^(R). Clone #56/36 which displayed two internal HaeIII siteswas digested with this enzyme to produce a probe to identity PCRpositive clones.

After amplification the PCR products were checked on a 1.2% (w/v)agarose gel. Cleaning up of the PCR products included aphenol-chloroform extraction followed by further purification by HPLC(GEN-PAC FAX column, Waters) and finally by using the MERMAID orGENECLEAN II kit, BIO 101, Inc) as appropriate. After these purificationsteps, the amplified cDNA was then digested with EcoRI and KpnI forseries #56 clones and with XhoI and KpnI for series #72 clones. A finalphenol-chloroform extraction preceded the ligation into pUC 18(series=56 clones) or into pBluescript^(R) (series=72 clones).

All the clones obtained were smaller that the 860 base pairs to beexpected if they possessed a complete V_(HH) and C_(H) 1 region. Partialsequence data corresponding to the N-terminal of the V_(HH) regionreveals that out of 20 clones, 3 were identical and possibly notindependent. The sequences obtained ressemble the human subgroup III andthe murine subgroups IIIa and IlIb (Table 2).

Clones corresponding to two different sets of C_(H) 2 protein sequenceswere obtained. A first set of sequences (=72/41) had a N-terminal C_(H)2 region identical to the one obtained by protein sequencing of the 28Kd papain fragments of the γ³ heavy chain, a short hinge regioncontaining 3 cysteines and a variable region corresponding to theframework (FR4) residues encoded by the J minigenes adjoining the hinge.The C_(H) 1 domain is entirely lacking. This cDNA corresponds to the γ3chain (Table 4).

In one closely related sequence (#72/1) the proline in position 259 isreplaced by threonine.

The sequence corresponding to the C_(H) 3 and the remaining part of theC_(H) 2 was obtained by PCR of the cDNA using as KpnI primer a poly T inwhich a KpnI restriction site had been inserted at the 5' end. The totalsequence of the γ3 chain corresponds to a molecular weight (MW) which isin good agreement with the data obtained from SDS PAGE electrophoresis.

The sequence of this γ3 chain presents similarities with other γ chainsexcept that it lacks the C_(H) 1 domain, the V_(HH) domain beingadjacent to the hinge.

One or all three of the cysteines could be probably responsible forholding the two γ3 chains together.

These results have allowed us to define a model for the IgG3 moleculebased on sequence and papain cleavage (FIG. 5).

Papain can cleave the molecule on each side of the hinge disulfides andalso between C_(H) 2 and C_(H) 3. Under non reducing conditions theV_(HH) domains of IgG3 can be isolated as disulfide linked dimer or asmonomer depending on the site of papain cleavage.

A second set of clones #72/29 had a slightly different sequence for theC_(H) 2 and was characterized by a very long hinge immediately precededby the variable domain. This hinge region has 3 cysteines at itsC-terminal end in a sequence homologeous to the γ3 hinge. Such secondset of clones could represent the IgG2 subclass. For the constant partof the γ3 and also for the putative γ2, most clones are identicalshowing the γ2 or γ3 specific sequences. A few clones such as #72/1however show minor differences. For instance in the case of clones #72/1two nucleotide differences are detected.

Several V_(HH) regions cDNA's have now been totally or partiallysequenced with the exception of a short stretch at the N-terminal endwhich is primer derived.

Upon translation the majority shows the characteristic heavy chain Ser₂₁Cys₂₂ and Tyr₉₀ Tyr₉₁ Cys₉₂ sequences, of the intra V_(HH) regiondisulfide bridge linking residues 22 and 92. All these clones have asequence corresponding to the framework 4 (FR4) residues of the variableregion immediately preceding the postulated hinge sequence (Table 3).This sequence is generated by the J minigenes and is in the majority ofcases similar to the sequence encoded by the human and murine sminigenes. The sequence length between region CYS₉₂ and the C-terminalend of the V_(HH) regions is variable and, in the sequences determined,range from 25 to 37 amino-acids as one might expect from therearrangements of J and D minigenes varying in length.

Several important questions are raised by the sole existence of theseheavy chain immunoglobulins in a non pathological situation. First ofall, are they bonafide antibodies? The heavy chain immunoglobulinsobtained from trypanosome infected camels react with a large number ofparasite antigens as shown in part I of these examples. This impliesthat the camelid immune system generates an extensive number of bindingsites composed of single V_(HH) domains. This is confirmed by thediversity of the V_(HH) regions of the heavy chain immunogobulinsobtained by PCR.

The second question is "how are they secreted ?". The secretion ofimmunoglobulin heavy chains composing four-chain model immunoglobulinsdoes not occur under normal conditions. A chaperoning protein, the heavychain binding protein, or BIP protein, prevents heavy chains from beingsecreted. It is only when the light chain displaces the BIP protein inthe endoplasmatic reticulum that secretion can occur (13).

The heavy chain dimer found in the serum of human or mice with theso-called "heavy chain disease" lack the C_(H) 1 domains thought toharbour the BIP site (14). In the absence of thi domain the BIP proteincan no longer bind and prevent the transport of the heavy chains.

The presence in camels of a IgG1 class composed of heavy and lightchains making up between 25% and 50% of the total IgG molecules alsoraises the problem as to how maturation and class switching occurs andwhat the role of the light chain is. The camelid light chain appearsunusually large and heterogeneous when examined in SDS PAGE.

The largest dimension of an isolated domain is 40 Å and the maximumattainable span between binding sites of a conventional IgG with C_(H) 1and V_(HH) will be of the order of 160 Å (2V_(HH) +2C_(H) 1) (19). Thedeletion of C_(H) 1 domain in the two types of heavy chain antibodiesdevoid of light chains, already sequenced has, as a result, amodification of this maximum span (FIG. 6). In the IgG3 the extremedistance between the extremities of the V_(HH) regions will be of theorder of 80 Å (2V_(HH)). This could be a severe limitation foragglutinating or cross linking. In the IgG2 this is compensated by theextremely long stretch of hinge, composed of a 12-fold repeat of thesequence Pro-X (where X is Gln, Lys or Glu) and located N-terminal tothe hinge disulfide bridges. In contrast, in the human IgG3, the verylong hinge which also apparently arose as the result of sequenceduplication does not contribute to increase the distance spanning thetwo binding sites as this hinge is inter-spersed with disulfide bridges.

The single V_(HH) domain could also probably allow considerablyrotational freedom of the binding site versus the Fc domain.

Unlike myeloma heavy chains which result probably from C_(H) 1 deletionin a single antibody producing cell, or heavy chain antibodies producedby expression cloning(15); the camelid heavy chain antibodies (devoid oflight chains) have emerged in a normal immunological environment and itis expected that they will have undergone the selective refinement inspecificity and affinity accompanying B cell maturation.

Expression and purification of the camel V_(HH) 21 (DR21 on FIG. 7)protein from E. coli

The clones can be expressed in several types of expression vectors. Asan example using a commercially available vector Immuno PBS (Huse et al:Science (1989) 246, 1275), clones produced in Bluescript® according tothe above described procedure, have been recovered by PCR using the sameXhoI containing 5' primer and a new 3' primer, corresponding to residues113-103 in the framework of the immunoglobulins, in which an Spe sitehas been constructed: TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO:51. This procedure allowed the cloning of the V_(HH) in the Xho/Spe siteof the Immuno PBS vector. However, the 3' end of the gene was not inphase with the identification "tag" and the stop codon of the vector. Toachieve this, the construct was cut with Spe and the 4 base overhangswere filled in, using the Klenow fragment after which the vector wasreligated.

The expression vector plasmid ipBS (immunopBS) (Stratacyte) contains apel B leader sequence which is used for immunoglobulin chain expressionin E. coli under the promotor pLAC control, a ribosome binding site, andstop codons. In addition, it contains a sequence for a c-terminaldecapeptide tag.

E. coli JM101 harboring the ipBS-V_(HH) 21 plasmid was grown in 1 l ofTB medium with 100 μg/ml ampicillin and 0.1% glucose at 32° C.Expression was induced by the addition of 1 mM 1 PTG (finalconcentration) at an OD₅₅₀ of 1.0. After overnight induction at 28° C.,the cells were harvested by centrifugation at 4.000 g for 10 min (4° C.)and resuspended in 10 ml TES buffer (0.2 M Tris-HCL pH 8.0, 0.5 mM EDTA,0.5 M sucrose). The suspension was kept on ice for 2 hours. Periplasmicproteins were removed by osmotic shock by addition of 20 ml TES bufferdiluted 1:4 v/v with water, kept on ice for one hour and subsequentlycentrifugated at 12.000 g for 30 min. at 4° C. The supernatantperiplasmic fraction was dialysed against Tris-HCl pH 8.8, NaCl 50 mM,applied on a fast Q Sepharose flow (Pharmacia) column, washed with theabove buffer prior and eluted with a linear gradient of 50 mM to 1 MNaCl in buffer.

Fractions containing the V_(HH) protein were further purified on aSuperdex 75 column (Pharmacia) equilibrated with PBS buffer (0.01 Mphosphate pH 7.2, 0.15 M NaCl). The yield of purified V_(HH) proteinvaries from 2 to 5 mg/l cell culture.

Fractions were analyzed by SDS-PAGE(I). Positive identification of thecamel V_(HH) antibody fragment was done by western Blot analysis usingantibody raised in rabbits against purified camel IgGH₃ and ananti-rabbit IgG-alkaline phosphatase conjugate (II).

As protein standards (Pharmacia) periplasmic proteins prepared from 1 mlof IPTG-induced JM101/ipBS V_(HH) 21 were used. FIG. 8 shows:C,D:fractions from fast S Sepharose column chromatography (C:Eluted at650 mM NaCl D:Eluted at 700 mM NaCl) E,F:fractions from Superdex 75column chromatography.

As can be seen, the major impurity is eliminated by ionexchangechromatography and the bulk of the remaining impurities are eliminatedby gel filtration.

                                      TABLE 1                                     __________________________________________________________________________    Comparison of the N terminal Camel C.sub.H 2 and C.sub.H 3 sequences with     the translated                                                                 cDNA sequences of Camel immunoglolins and with the corresponding human       γ                                                                         sequences. (Numbering according to Kabat et al (1987)(7).                   __________________________________________________________________________                      250                 260                 270                   Camel γ3 28Kd                                                           L P G G P S V F V F P P K P K D V L S I X G X P SEQ ID NO:54 - -                                                        - Clone# 72/1                       L P G G P S V F V F P T K P K D V L S I S G R P SEQ ID NO:55 - -                                                        - Clone# 72/4                       L P G G P S V F V F P P K P K D V L S I S G R P SEQ ID NO:56 - -                                                        - Clone# 72/29                      L L G G P S V F I F P P K P K D V L S I S G R P SEQ ID NO:57 - -                                                        - Human γ1γ3            L L G G P S V F L F P P K P K D T L M I S R T P SEQ ID NO:112 - -                                                       - C.sub.H 2 γ2                V A - G P S V F L F P P K P K D T L M I S R T P SEQ ID NO:113 - -                                                       -  γ4                         F L G G P S V F L F P P K P K D T L M I S R T P SEQ ID NO:114 - -                                                       - C.sub.H 2|C.sub.H                                                3                                      360                370                                                       Camel γ3 14Kd                                                           K|G Q T R E P Q V Y T L A P X R L E L SEQ ID NO:54 - -                                                         - Human γ1                    K|G Q P R E P Q V Y T L P P S R D E L SEQ ID NO:115 - -                                                        - C.sub.H 2/C.sub.H 3                                                       γ2,γ3                     K|G Q P R E P Q V Y T L P P S R E E M SEQ ID NO:116 - -                                                        -  γ4                         K|G Q P R E P Q V Y T L P P S Q E E M SEQ ID NO:117 - -            __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    A Comparison of N Terminal Fr 1 regions of Camel V.sub.HH with a Human        V.sub.H  III subgroup                                                           protein and a mouse V.sub.H  IIIA subgroup protein.                           The invariable subgroup specific residues are grayed.                       __________________________________________________________________________                     10                  20                  30                      Primer Derived G G S V Q T G G S L R L S C E I S G L T F D # 72/4 SEQ                                                  ID NO:1                              -                 G G S V Q T G G S L R L S C A V S G F S F S # 72/3                                                   SEQ ID NO:2                          -                 G G S E Q G G G S L R L S C A I S G Y T Y G # 72/7                                                   SEQ ID NO:3                          -                 G G S V Q P G G S L T L S C T V S G A T Y S # 72/17                                                  SEQ ID NO:4                          -                 G G S V Q A G G S L R L S C T G S G F P Y S # 72/18                                                  SEQ ID NO:5                          - D V Q L V A S G G G S V G A G G S L R L S C T A S G D S F S # 72/2                                                   SEQ ID NO:58                         - E V K L V E S G G G L V E P G G S L R L S C A T S G F T F S Mouse                                                    V.sub.H III.sub.A  SEQ ID                                                     NO:118                               -   E V Q L L S G G G L V Q P G G S L R L S C A A S G F T F S Human                                                    V.sub.H III SEQ ID NO:119         __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Comparison of some Framework 4 residues found in the Camel V.sub.HH            region with the Framework 4 residues corresponding to the                     consensus region of the Human and Mouse J minigenes.                         FrameWork 4                                                                   __________________________________________________________________________                                 J Genes                                                                        Human W G Q G T L V T V S S SEQ ID NO:9 J1,                                  J4, J5                                              - W G R G T L V T V S S SEQ ID NO:130J2                                       - W G Q G T T V T V S S SEQ ID NO:120J6                                       - W G Q G T M V T V S S SEQ ID NO:121J3                                       -  - MurineW G Q G T T L T V S S SEQ ID NO:122J1                              - W G Q G T L V T V S S SEQ ID NO:9J2                                         - W G Q G T S V T V S A SEQ ID NO:123J3                                       - W G A G T T V T V S S SEQ ID NO:124J4                                       -  - cDNA Clones                                                              - CamelW G Q G T Q V T V S S SEQ ID NO:8Clones                                - W G Q G T Q V T V S S SEQ ID NO:8# 72/19 = # 72/3                           - W G Q G T L V T V S S SEQ ID NO:91 Clone                                    - W G R G T Q V T V S S SEQ ID NO:59# 72/24                                   - W G Q G T H V T V S S SEQ ID NO:60# 72/21                                   - W G Q G I Q V T A S S SEQ ID NO:61# 72/16                                __________________________________________________________________________

    TABLE 4       -   d y y g s s - - - - - - - y - - f - - - - - d v W G A G T T V T S     S SEQ ID NO:125 Mouse V.sub.H      .sbsb.III sequence                           95        100 a b c d e f     g h i j k - - - - 101     105      110                                        1  a l q p g g y c g y g x  - - - - -       - -  - - c l W G Q G T Q V T V S      S SEQ ID NO:13                                       2  v s l m d r i s q h - - - - - - - -       - - - - g c R G Q G T Q V T V S      L SEQ ID NO:14                                      3  v p a h l g p g s i l d l k k y - -     - - - - k y W G Q G T Q V T V S      S SEQ ID NO:15                                    4  f c y s t a g d g g s g e - - - - - -     - - - m y W G Q G T Q V T V S      S SEQ ID NO:16                                  7  e l s g g s c e l p l l f - - - - - - -     - - d y W G Q G T Q V T V S      S SEQ ID NO:17                                9  d w k y w t c g a q t g g y f - - - - - -     - g q W G Q G A Q V T V S      S SEQ ID NO:18                              11 r l t e m g a c d a r w a t l a t r t f a y     n y W G Q G T Q V T V S      S SEQ ID NO:19 Random Sample                            13 q k k d r t r w a e p r e w- - - - - - - -  n     n W G Q G T Q V T A S      S SEQ ID NO:20                          16 g s r f s s p v g a t s r l e s - s d y - - n y     W G Q G I Q V T A S      S SEQ ID NO:21                        17 a d p s i y y s i l x i e y - - - - - - - - k y W     G Q G T Q V T V S      S SEQ ID NO:22 18 different camel                      18 d s p c y m p t m p a p p i r d s f g w - - d d F G     O G T O V T V S S SEQ ID NO:23 V.sub.HH      region                                             19 t s s t y w y c t t a p y - -       - - - - - - - n v W G Q G T Q V T V S      S SEQ ID NO:24                                            20 t e i e w y g c n l r t t f -     - - - - - - - t r W G Q G T Q V T A S      S SEQ ID NO:25                                          21 n g l a g g w y l d p n y w l s     v g a y - - a i W G Q G T H V T V S      S SEQ ID NO:26                                        24 r l t e m g a c d a r w a t l a t     r t f a y n y W G R G T Q V T V S      S SEQ ID NO:27                                      25 d g w t r k e g g i g l p w s v q c     e d g y n y W G Q G T Q V T V S      S SEQ ID NO:28                                    27 d s y p c h i i - - - - - - - - - - -     - - - d v W G Q G T Q V T V S      S SEQ ID NO:29                                  29 v e y p i a d m c s - - - - - - - - - -     - - r y G D P G T Q V T V S      S SEQ ID NO:30      ##STR1##

                                      TABLE 5                                     __________________________________________________________________________                                                            #STR2##                  -                                                                                                                                  #STR3##                  -                                                                                                                                  #STR4##               __________________________________________________________________________

REFERENCES

1. Ward, E. S., Gussow, D., Griffits, A. D., Jones, P. T. and Winter G.Nature 341, 544-546 (1989).

2. Ungar-Waron H., Eliase E., Gluckman A. and Trainini Z. Isr. J. Vet.Med., 43, 198-203 (1987).

3. Bajyana Songa E. and Hamers R., Ann. Soc. Belge Med. trop., 68,233-240 (1988).

4. Edelman, G. M., Olins, D. E., Gally, J. A. and Zinder, N. D., Proc.Nat. Acad. Sc., 50, 753 (1963).

5. Franek F. and Nezlin, R. S., Biokhimiya, 28, 193 (1963).

6. Roitt, I. M., Brostof, J. and Male, D. K., Immunology, Gower Med.Pub. London. New-York, p.9.2. (1985).

7. Schiffer, M., Girling, R. L., Ely, K. R. and Edmundson, B.,Biochemistry, 12, 4620-4631 (1973).

8. Fleischman J. B., Pain R. H. and Porter R. R., Arch. Biochem.Biophys, Suppl. 1, 174 (1962).

9. Roholt O., Onoue K. and Pressman D., PNAS 51, 173-178 (1964).

10. Seligmann M., Mihaesco E., Preud'homme J. L., Danon F. and Brouet J.C., Immunological Rev., 48, 145-167 (1979).

11. Henderschot L., Bole D., Kohler G. and Kearney J. F., The Journal ofCell Biology, 104, 761-767 (1987).

12. Henderschot L. M., The Journal of Cell Biology, 111, 829-837 (1990).

13. Hamers-Casterman, C., E. Wittouck, W. Van der Loo and R. Hamers,Journal of Immunogenetics, 6, 373-381 (1979).

14. Applied Biosystems--Ethanol Precipitation of Electro ElutedElectrodialysed Sample. Issue n° 27.

15. Maniatis, T. E. F. Fritsch and J. Sambrook, Molecular Cloning. ALaboratory Manual (1988).

16. Sastry et al., PNAS, 86, 5728, (1989).

17. Sanger, F., S. Nicklen and A. R. Coulson, Proc. Natl. Acad. Sci.,U.S.A., 74, 5463-5467 (1977).

18. Kabat E. A., Tai Te Wu, M. Reid-Miller, H. M. Perry and K. S.Gottesman, U.S. Dpt of Health and Human Services, Public Health Service,National Institutes of Health (1987).

19. Valentine, R. C. and N. M. Geen, J. M. B., 27, 615-617 (1967).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 130                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:1:                        - -      Gly Gly Ser Val Gln Thr Gly Gly - # Ser Leu Arg Leu Ser Cys       Glu Ile                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Gly Leu Thr Phe Asp                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:2:                        - -      Gly Gly Ser Val Gln Thr Gly Gly - # Ser Leu Arg Leu Ser Cys       Ala Val                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Gly Phe Ser Phe Ser                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:3:                        - -      Gly Gly Ser Glu Gln Gly Gly Gly - # Ser Leu Arg Leu Ser Cys       Ala Ile                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Gly Tyr Thr Tyr Gly                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:4:                        - -      Gly Gly Ser Val Gln Pro Gly Gly - # Ser Leu Thr Leu Ser Cys       Thr Val                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Gly Ala Thr Tyr Ser                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:5:                        - -      Gly Gly Ser Val Gln Ala Gly Gly - # Ser Leu Arg Leu Ser Cys       Thr Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Gly Phe Pro Tyr Ser                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:6:                        - -      Gly Gly Ser Val Gln Ala Gly Gly - # Ser Leu Arg Leu Ser Cys       Val Ala                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gly Phe Gly Thr Ser                                                                  20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 1                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:7:                        - -      Gly Gly Ser Val Gln Ala Gly Gly - # Ser Leu Arg Leu Ser Cys       Val Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Phe Ser Pro Ser Ser                                                                  20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:8:                        - -      Trp Gly Gln Gly Thr Gln Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:9:                        - -      Trp Gly Gln Gly Thr Leu Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:10:                       - -      Trp Gly Gln Gly Ala Gln Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:11:                       - -      Trp Gly Gln Gly Thr Gln Val Thr - # Ala Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= FRAMEWORK 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:12:                       - -      Arg Gly Gln Gly Thr Gln Val Thr - # Val Ser Leu                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:13:                       - -      Ala Leu Gln Pro Gly Gly Tyr Cys - # Gly Tyr Gly Xaa Cys Leu       Trp Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln Gly Thr Gln Val Thr Val Ser - # Ser                                              20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:14:                       - -      Val Ser Leu Met Asp Arg Ile Ser - # Gln His Gly Cys Arg Gly       Gln Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Gln Val Thr Val Ser Leu                                                          20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:15:                       - -      Val Pro Ala His Leu Gly Pro Gly - # Ala Ile Leu Asp Leu Lys       Lys Tyr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Lys Tyr Trp Gly Gln Gly Thr Gln - # Val Thr Val Ser Ser                             20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:16:                       - -      Phe Cys Tyr Ser Thr Ala Gly Asp - # Gly Gly Ser Gly Glu Met        Tyr Trp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gly Gln Gly Thr Gln Val Thr Val - # Ser Ser                                          20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:17:                       - -      Glu Leu Ser Gly Gly Ser Cys Glu - # Leu Pro Leu Leu Phe Asp       Tyr Trp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gly Gln Gly Thr Gln Val Thr Val - # Ser Ser                                          20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:18:                       - -      Asp Trp Lys Tyr Trp Thr Cys Gly - # Ala Gln Thr Gly Gly Tyr       Phe Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln Trp Gly Gln Gly Ala Gln Val - # Thr Val Ser Ser                                  20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:19:                       - -      Arg Leu Thr Glu Met Gly Ala Cys - # Asp Ala Arg Trp Ala Thr       Leu Ala                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Arg Thr Phe Ala Tyr Asn Tyr - # Trp Gly Gln Gly Thr Gln       Val Thr                                                                                          20 - #                 25 - #                 30             - -      Val Ser Ser                                                                      35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:20:                       - -      Gln Lys Lys Asp Arg Thr Arg Trp - # Ala Glu Pro Arg Glu Trp        Asn Asn                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Trp Gly Gln Gly Thr Gln Val Thr - # Ala Ser Ser                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:21:                       - -      Gly Ser Arg Phe Ser Ser Pro Val - # Gly Ser Thr Ser Arg Leu       Glu Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Asp Tyr Asn Tyr Trp Gly Gln - # Gly Thr Gln Val Thr Ala       Ser Ser                                                                                          20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:22:                       - -      Ala Asp Pro Ser Ile Tyr Tyr Ser - # Ile Leu Xaa Ile Glu Tyr        Lys Tyr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Trp Gly Gln Gly Thr Gln Val Thr - # Val Ser Ser                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:23:                       - -      Asp Ser Pro Cys Tyr Met Pro Thr - # Met Pro Ala Pro Pro Ile       Arg Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Phe Gly Trp Asp Asp Phe Gly - # Gln Gly Thr Gln Val Thr       Val Ser                                                                                          20 - #                 25 - #                 30             - -      Ser                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:24:                       - -      Thr Ser Ser Phe Tyr Trp Tyr Cys - # Thr Thr Ala Pro Tyr Asn        Val Trp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gly Gln Gly Thr Gln Val Thr Val - # Ser Ser                                          20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:25:                       - -      Thr Glu Ile Glu Trp Tyr Gly Cys - # Asn Leu Arg Thr Thr Phe       Thr Arg                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Trp Gly Gln Gly Thr Gln Val Thr - # Val Ser Ser                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:26:                       - -      Asn Gln Leu Ala Gly Gly Trp Tyr - # Leu Asp Pro Asn Tyr Trp       Leu Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Gly Ala Tyr Ala Ile Trp Gly - # Gln Gly Thr His Val Thr       Val Ser                                                                                          20 - #                 25 - #                 30             - -      Ser                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:27:                       - -      Arg Leu Thr Glu Met Gly Ala Cys - # Asp Ala Arg Trp Ala Thr        Leu Ala                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Arg Thr Phe Ala Tyr Asn Tyr - # Trp Gly Arg Gly Thr Gln       Val Thr                                                                                          20 - #                 25 - #                 30             - -      Val Ser Ser                                                                      35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:28:                       - -      Asp Gly Trp Thr Arg Lys Glu Gly - # Gly Ile Gly Leu Pro Trp        Ser Val                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln Cys Glu Asp Gly Tyr Asn Tyr - # Trp Gly Gln Gly Thr Gln       Val Thr                                                                                          20 - #                 25 - #                 30             - -      Val Ser Ser                                                                      35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:29:                       - -      Asp Ser Tyr Pro Cys His Leu Leu - # Asp Val Trp Gly Gln Gly        Thr Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Thr Val Ser Ser                                                                  20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:30:                       - -      Val Glu Tyr Pro Ile Ala Asp Met - # Cys Ser Arg Tyr Gly Asp       Pro Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Gln Val Thr Val Ser Ser                                                          20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..27                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:31:                       - -      Ala Pro Glu Leu Leu Gly Gly Pro - # Thr Val Phe Ile Phe Pro       Pro Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Thr - # Leu Thr Pro                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..27                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:32:                       - -      Ala Pro Glu Leu Pro Gly Gly Pro - # Ser Val Phe Val Phe Pro       Thr Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Arg Pro                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..27                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:33:                       - -      Ala Pro Glu Leu Pro Gly Gly Pro - # Ser Val Phe Val Phe Pro       Pro Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Arg Pro                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..27                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:34:                       - -      Ala Pro Glu Leu Leu Gly Gly Pro - # Ser Val Phe Ile Phe Pro       Pro Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Arg Pro                                      20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= CH3                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:35:                       - -      Gly Gln Thr Arg Glu Pro Gln Val - # Tyr Thr Leu Ala                      1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/label= CH3                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:36:                       - -      Gly Gln Thr Arg Glu Pro Gln Val - # Tyr Thr Leu Ala Pro Xaa       Arg Leu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Leu                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= hinge                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:37:                       - -      Gly Thr Asn Glu Val Cys Lys Cys - # Pro Lys Cys Pro                      1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..35                                                           (D) OTHER INFORMATION: - #/label= hinge                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:38:                       - -      Glu Pro Lys Ile Pro Gln Pro Gln - # Pro Lys Pro Gln Pro Gln       Pro Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Gln Pro Lys Pro Gln Pro Lys - # Pro Glu Pro Glu Cys Thr       Cys Pro                                                                                          20 - #                 25 - #                 30             - -      Lys Cys Pro                                                                      35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..28                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:39:                       - -      Ala Pro Glu Leu Leu Gly Gly Pro - # Ser Val Phe Val Phe Pro        Pro Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Xaa Pro Lys                                  20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..28                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:40:                       - -      Ala Pro Glu Leu Pro Gly Gly Pro - # Ser Val Phe Val Phe Pro       Thr Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Arg Pro Lys                                  20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..28                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:41:                       - -      Ala Pro Glu Leu Pro Gly Gly Pro - # Ser Val Phe Val Phe Pro       Pro Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Arg Pro Lys                                  20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..28                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:42:                       - -      Ala Pro Glu Leu Leu Gly Gly Pro - # Ser Val Phe Ile Phe Pro       Pro Lys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Lys Asp Val Leu Ser Ile Ser - # Gly Arg Pro Lys                                  20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:43:                       - -      Val Thr Val Ser Ser Gly Thr Asn - # Glu Val Cys Lys Cys Pro       Lys Cys                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Pro Ala Pro Glu Leu Pro Gly Gly - # Pro Ser Val Phe Val Phe       Pro                                                                                              20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..54                                                           (D) OTHER INFORMATION: - #/label= hinge                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:44:                       - -      Val Thr Val Ser Ser Glu Pro Lys - # Ile Pro Gln Pro Gln Pro        Lys Pro                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln Pro Gln Pro Gln Pro Gln Pro - # Lys Pro Gln Pro Lys Pro       Glu Pro                                                                                          20 - #                 25 - #                 30             - -      Glu Cys Thr Cys Pro Lys Cys Pro - # Ala Pro Glu Leu Leu Gly        Gly Pro                                                                                      35     - #             40     - #             45                  - -      Ser Val Phe Ile Phe Pro                                                      50                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: - #/label= hinge                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:45:                       - -      Ala Pro Glu Leu Pro Gly Gly Pro - # Ser Val Phe Val Phe Pro             1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:46:                       - -      Ala Pro Glu Leu Leu Gly Gly Pro - # Ser Val Phe Ile Phe Pro              1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:47:                       - - CGCCATCAAG GTAACAGTTG A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..17                                                          (D) OTHER INFORMATION: - #/label= XhoI site                                        /note= - #"RESTRICTION SITE"                                    - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:48:                       - - AGGTCCAGCT GCTCGAGTCT GG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..17                                                          (D) OTHER INFORMATION: - #/label= XhoI site                                        /note= - #"Restriction site"                                    - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:49:                       - - AGCTCCAGCT GCTCGAGTCT GG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 12..17                                                          (D) OTHER INFORMATION: - #/label= XhoI site                                        /note= - #"restriction site"                                    - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:50:                       - - AGGTCCAGCT TCTCGAGTCT GG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:51:                       - - TCTTAACTAG TGAGGAGACG GTGACCTG         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: - #/label= SpeI                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:52:                       - - CTAGTGCACC ACCATCACCA TCACTAATAG         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: Other                                                      (A) DESCRIPTION: DNA (s - #ynthetic)                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..30                                                           (D) OTHER INFORMATION: - #/note= "Sequence complementary to                        SEQ ID - #NO: 52"                                               - -     (ix) FEATURE:                                                                  (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 26..30                                                          (D) OTHER INFORMATION: - #/label= EcoRI                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:53:                       - - ACGTGGTGGT AGTGGTAGTG ATTATCTTAA         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..25                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 26..43                                                          (D) OTHER INFORMATION: - #/label= CH3                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:54:                       - -      Leu Pro Gly Gly Pro Ser Val Phe - # Val Phe Pro Pro Lys Pro        Lys Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Leu Ser Ile Xaa Gly Xaa Pro - # Lys Gly Gln Thr Arg Glu       Pro Gln                                                                                          20 - #                 25 - #                 30             - -      Val Tyr Thr Leu Ala Pro Xaa Arg - # Leu Glu Leu                                  35     - #             40                                         - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CH2                                              /note= - #"Clone #72/1"                                         - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:55:                       - -      Leu Pro Gly Gly Pro Ser Val Phe - # Val Phe Pro Thr Lys Pro        Lys Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Leu Ser Ile Ser Gly Arg Pro                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:56:                       - -      Leu Pro Gly Gly Pro Ser Val Phe - # Val Phe Pro Pro Lys Pro       Lys Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Leu Ser Ile Ser Gly Arg Pro                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CH2                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:57:                       - -      Leu Leu Gly Gly Pro Ser Val Phe - # Ile Phe Pro Pro Lys Pro       Lys Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Leu Ser Ile Ser Gly Arg Pro                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..30                                                           (D) OTHER INFORMATION: - #/label= Framework 1                                      /note= - #"CAMEL"                                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:58:                       - -      Asp Val Gln Leu Val Ala Ser Gly - # Gly Gly Ser Val Gly Ala       Gly Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Leu Arg Leu Ser Cys Thr Ala - # Ser Gly Asp Ser Phe Ser                         20 - #                 25 - #                 30              - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= Framework 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:59:                       - -      Trp Gly Arg Gly Thr Gln Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= Framework 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:60:                       - -      Trp Gly Gln Gly Thr His Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..11                                                           (D) OTHER INFORMATION: - #/label= Framework 4                        - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:61:                       - -      Trp Gly Gln Gly Ile Gln Val Thr - # Ala Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..14                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:62:                       - -      Ala Leu Gln Pro Gly Gly Tyr Cys - # Gly Tyr Gly Xaa Cys Leu              1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:63:                       - -      Val Ser Leu Met Asp Arg Ile Ser - # Gln His Gly Cys                      1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..18                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:64:                       - -      Val Pro Ala His Leu Gly Pro Gly - # Ala Ile Leu Asp Leu Lys        Lys Tyr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Lys Tyr                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus b - #actrianus                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:65:                       - -      Phe Cys Tyr Ser Thr Ala Gly Asp - # Gly Gly Ser Gly Glu Met       Tyr                                                                                  1             - #  5                - #   10               - #         15                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:66:                       - -      Glu Leu Ser Gly Gly Ser Cys Glu - # Leu Pro Leu Leu Phe Asp       Tyr                                                                                  1             - #  5                - #   10               - #         15                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..17                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:67:                       - -      Asp Trp Lys Tyr Trp Thr Cys Gly - # Ala Gln Thr Gly Gly Tyr       Phe Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:68:                       - -      Arg Leu Thr Glu Met Gly Ala Cys - # Asp Ala Arg Trp Ala Thr       Leu Ala                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Arg Thr Phe Ala Tyr Asn Tyr                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:69:                       - -      Gln Lys Lys Asp Arg Thr Arg Trp - # Ala Glu Pro Arg Glu Trp       Asn Asn                                                                              1             - #  5                - #   10               - #         15                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:70:                       - -      Gly Ser Arg Phe Ser Ser Pro Val - # Gly Ser Thr Ser Arg Leu       Glu Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Asp Tyr Asn Tyr                                                                  20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:71:                       - -      Ala Asp Pro Ser Ile Tyr Tyr Ser - # Ile Leu Xaa Ile Glu Tyr       Lys Tyr                                                                              1             - #  5                - #   10               - #         15                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:72:                       - -      Asp Ser Pro Cys Tyr Met Pro Thr - # Met Pro Ala Pro Pro Ile       Arg Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Phe Gly Trp Asp Asp                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..15                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:73:                       - -      Thr Ser Ser Phe Tyr Trp Tyr Cys - # Thr Thr Ala Pro Tyr Asn       Val                                                                                  1             - #  5                - #   10               - #         15                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..16                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:74:                       - -      Thr Glu Ile Glu Trp Tyr Gly Cys - # Asn Leu Arg Thr Thr Phe       Thr Arg                                                                              1             - #  5                - #   10               - #         15                                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..22                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:75:                       - -      Asn Gln Leu Ala Gly Gly Trp Tyr - # Leu Asp Pro Asn Tyr Trp       Leu Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Gly Ala Tyr Ala Ile                                                              20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:76:                       - -      Arg Leu Thr Glu Met Gly Ala Cys - # Asp Ala Arg Trp Ala Thr       Leu Ala                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Arg Thr Phe Ala Tyr Asn Tyr                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..24                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:77:                       - -      Asp Gly Trp Thr Arg Lys Glu Gly - # Gly Ile Gly Leu Pro Trp       Ser Val                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln Cys Glu Asp Gly Tyr Asn Tyr                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:78:                       - -      Asp Ser Tyr Pro Cys His Leu Leu - # Asp Val                              1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= VH                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..12                                                           (D) OTHER INFORMATION: - #/label= CDR3                               - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:79:                       - -      Val Glu Tyr Pro Ile Ala Asp Met - # Cys Ser Arg Tyr                      1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:80:                       - -      Glu Val Gln Leu Val Glu Ser Gly - # Gly Gly Leu Val Gln Pro       Gly Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Leu Arg Leu Ser Cys Ala Ala - # Ser Gly                                          20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Camelus d - #romedarius                                - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:81:                       - -      Trp Val Arg Gln Ala Pro Gly Lys - # Gly Leu Glu Trp Val Ser             1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:82:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:82:                       - -      Arg Phe Thr Ile Ser Arg Asp Asn - # Ser Lys Asn Thr Leu Tyr        Leu Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Met Asn Ser Leu Arg Ala Glu Asp - # Thr Ala Val Tyr Tyr Cys       Ala Arg                                                                                          20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:83:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:83:                       - -      Trp Gly Gln Gly Thr Leu Val Thr - # Val Ser Ser Gly Thr Asn        Glu Val                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Cys Lys Cys Pro Lys Cys Pro Ala - # Pro Glu Leu Pro Gly Gly       Pro Ser                                                                                          20 - #                 25 - #                 30             - -      Val Phe Val Phe Pro                                                              35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:84:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:84:                       - -      Gly Gly Ser Val Gln Gly Gly Gly - # Ser Leu Arg Leu Ser Cys        Ala Ile                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Gly                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:85:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:85:                       - -      Trp Phe Arg Glu Gly Pro Gly Lys - # Glu Arg Glu Gly Ile Ala             1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:86:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:86:                       - -      Arg Phe Thr Ile Ser Gln Asp Ser - # Thr Leu Lys Thr Met Tyr        Leu Leu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Met Asn Asn Leu Lys Pro Glu Asp - # Thr Gly Thr Tyr Tyr Cys       Ala Ala                                                                                          20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:87:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 60 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:87:                       - -      Trp Gly Gln Gly Thr Gln Val Thr - # Val Ser Ser Glu Pro Lys        Ile Pro                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Gln Pro Gln Pro Lys Pro Gln Pro - # Gln Pro Gln Pro Gln Pro       Lys Pro                                                                                          20 - #                 25 - #                 30             - -      Gln Pro Lys Pro Glu Pro Glu Cys - # Thr Cys Pro Lys Cys Pro        Ala Pro                                                                                      35     - #             40     - #             45                  - -      Glu Leu Leu Gly Gly Pro Ser Val - # Phe Ile Phe Pro                          50         - #         55         - #         60                      - -  - - (2) INFORMATION FOR SEQ ID NO:88:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:88:                       - -      Gly Gly Ser Val Gln Ala Gly Gly - # Ser Leu Arg Leu Ser Cys       Ala Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Ser                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:89:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:89:                       - -      Trp Tyr Arg Gln Ala Pro Gly Lys - # Glu Arg Glu Phe Val Ser             1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:90:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:90:                       - -      Arg Phe Thr Ile Ser Gln Asp Ser - # Ala Lys Asn Thr Val Tyr        Leu Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Met Asn Ser Leu Lys Pro Glu Asp - # Thr Ala Met Tyr Tyr Cys       Lys Ile                                                                                          20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:91:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:91:                       - -      Trp Gly Gln Gly Thr Gln Val Thr - # Val Ser Ser Gly Thr Asn        Glu Val                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Cys Lys Cys Pro Lys Cys Pro Ala - # Pro Glu Leu Pro Gly Gly       Pro Ser                                                                                          20 - #                 25 - #                 30             - -      Val Phe Val Phe Pro                                                              35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:92:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 400 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:92:                       - - CTCGAGTCTG GGGGAGGATC GGTGCAGGCT GGAGGGTCTC TGAGACTCTC GT -             #GCGCAGCC     60                                                                 - - TCTGGATACA GTAATTGTCC CCTCACTTGG AGCTGGTATC GCCAGTTTCC AG -            #GAACGGAG    120                                                                 - - CGCGAGTTCG TCTCCAGTAT GGATCCGGAT GGAAATACCA AGTACACATA CT -            #CCGTGAAG    180                                                                 - - GGCCGCTTCA CCATGTCCCG AGGCAGCACC GAGTACACAG TATTTCTGCA AA -            #TGGACAAT    240                                                                 - - CTGAAACCTG AGGACACGGC GATGTATTAC TGTAAAACAG CCCTACAACC TG -            #GGGGTTAT    300                                                                 - - TGTGGGTATG GGTANTGCCT CTGGGGCCAG GGGACCCAGG TCACCGTCTC CT -            #CACTAGTT    360                                                                 - - ACCCGTACGA CGTTCCGGAC TACGGTTCTT AATAGAATTC     - #                      - #   400                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:93:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 391 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:93:                       - - CTCGAGTCTG GGGGAGGCTC GGTGCAGGCT GGAGGGTCTC TGAGACTCTC CT -             #GTGCATCT     60                                                                 - - TCTTCTAAAT ATATGCCTTG CACCTACGAC ATGACCTGGT ACCGCCAGGC TC -            #CAGGCAAG    120                                                                 - - GAGCGCGAAT TTGTCTCAAG TATAAATATT GATGGTAAGA CAACATACGC AG -            #ACTCCGTG    180                                                                 - - AAGGGCCGAT TCACCATCTC CCAAGACAGC GCCAAGAACA CGGTGTATCT GC -            #AGATGAAC    240                                                                 - - AGCCTGAAAC CTGAGGACAC GGCGATGTAT TACTGTAAAA TAGATTCGTA CC -            #CGTGCCAT    300                                                                 - - CTCCTTGATG TCTGGGGCCA GGGGACCCAG GTCACCGTCT CCTCACTAGT TA -            #CCCGTACG    360                                                                 - - AGCTTCCGGA CTACGGTTCT TAATAGAATT C        - #                  - #             391                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:94:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 443 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:94:                       - - CAGGTGAAAC TGCTCGAGTC TGGAGGAGGC TCGGTGCAGA CTGGAGGATC TC -             #TGAGACTC     60                                                                 - - TCCTGTGCAG TCTCTGGATT CTCCTTTAGT ACCAGTTGTA TGGCCTGGTT CC -            #GCCAGGCT    120                                                                 - - TCAGGAAAGC AGCGTGAGGG GGTCGCAGCC ATTAATAGTG GCGGTGGTAG GA -            #CATACTAC    180                                                                 - - AACACATATG TCGCCGAGTC CGTGAAGGGC CGATTCGCCA TCTCCCAAGA CA -            #ACGCCAAG    240                                                                 - - ACCACGGTAT ATCTTGATAT GAACAACCTA ACCCCTGAAG ACACGGCTAC GT -            #ATTACTGT    300                                                                 - - GCGGCGGTCC CAGCCCACTT GGGACCTGGC GCCATTCTTG ATTTGAAAAA GT -            #ATAAGTAC    360                                                                 - - TGGGGCCAGG GGACCCAGGT CACCGTCTCC TCACTAGCTA GTTACCCGTA CG -            #ACGTTCCG    420                                                                 - - GACTACGGTT CTTAATAGAA TTC           - #                  - #                   443                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:95:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 433 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:95:                       - - CTCGAGTCTG GGGGAGGGTC GGTGCAGGCT GGAGGGTCTC TGAGACTCTC CT -             #GTAATGTC     60                                                                 - - TCTGGCTCTC CCAGTAGTAC TTATTGCCTG GGCTGGTTCC GCCAGGCTCC AG -            #GGAGGGAG    120                                                                 - - CGTGAGGGGG TCACAGCGAT TAACACTGAT GGCAGTATCA TATACGCAGC CG -            #ACTCCGTG    180                                                                 - - AAGGGCCGAT TCACCATCTC CCAAGACACC GCCAAGGAAA CGGTACATCT CC -            #AGATGAAC    240                                                                 - - AACCTGCAAC CTGAGGATAC GGCCACCTAT TACTGCGCGG CAAGACTGAC GG -            #AGATGGGG    300                                                                 - - GCTTGTGATG CGAGATGGGC GACCTTAGCG ACAAGGACGT TTGCGTATAA CT -            #ACTGGGGC    360                                                                 - - CGGGGGACCC AGGTCACCGT CTCCTCACTA GTTACCCGTA CGACGTTCCG GA -            #CTACGGTT    420                                                                 - - CTTAATAGAA TTC              - #                  - #                      - #     433                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:96:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 449 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:96:                       - - CAGGTGAAAC TGCTCGAGTC TGGGGGAGGG TCGGTGCAGG CTGGAGGGTC TC -             #TGAGACTC     60                                                                 - - TCCTGTAATG TCTCTGGCTC TCCCAGTAGT ACTTATTGCC TGGGCTGGTT CC -            #GCCAGGCT    120                                                                 - - CCAGGGAAGG AGCGTGAGGG GGTCACAGCG ATTAACACTG ATGGCAGTGT CA -            #TATACGCA    180                                                                 - - GCCGACTCCG TGAAGGGCCG ATTCACCATC TCCCAAGACA CCGCCAAGAA AA -            #CGGTATAT    240                                                                 - - CTCCAGATGA ACAACCTGCA ACCTGAGGAT ACGGCCACCT ATTACTGCGC GG -            #CAAGACTG    300                                                                 - - ACGGAGATGG GGGCTTGTGA TGCGAGATGG GCGACCTTAG CGACAAGGAC GT -            #TTGCGTAT    360                                                                 - - AACTACTGGG GCCGGGGGAC CCAGGTCACC GTCTCCTCAC TAGCTAGTTA CC -            #CGTACGAC    420                                                                 - - GTTCCGGACT ACGGTTCTTA ATAGAATTC         - #                  - #               449                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:97:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 424 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:97:                       - - CTCGAGTCTG GAGGAGGCTC GGCGCAGGCT GGAGGATCTC TGAGACTCTC CT -             #GTGCAGCC     60                                                                 - - CACGGGATTC CGCTCAATGG TTACTACATC GCCTGGTTCC GTCAGGCTCC TG -            #GGAAGGGG    120                                                                 - - CGTGAGGGGG TCGCAACAAT TAATGGTGGT CGCGACGTCA CATACTACGC CG -            #ACTCCGTG    180                                                                 - - ACGGGCCGAT TTACCATCTC CCGAGACAGC CCCAAGAATA CGGTGTATCT GC -            #AGATGAAC    240                                                                 - - AGCCTGAAAC CTGAGGACAC GGCCATCTAC TTCTGTGCAG CAGGCTCGCG TT -            #TTTCTAGT    300                                                                 - - CCTGTTGGGA GCACTTCTAG ACTCGAAAGT AGCGACTATA ACTATTGGGG CC -            #AGGGGATC    360                                                                 - - CAGGTCACCG TCACCTCACT AGTTACCCGT ACGACGTTCC GGACTACGGT TC -            #TTAATAGA    420                                                                 - - ATTC                 - #                  - #                  - #                424                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:98:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 415 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:98:                       - - CTCGAGTCTG GAGGAGGCTC GGTTCAGGCT GGAGGGTCCC TTAGACTCTC CT -             #GTGCAGCC     60                                                                 - - TCTGACTACA CCATCACTGA TTATTGCATG GCCTGGTTCC GCCAGGCTCC AG -            #GGAAGGAG    120                                                                 - - CGTGAATTGG TCGCAGCGAT TCAAGTTGTC CGTAGTGATA CTCGCCTCAC AG -            #ACTACGCC    180                                                                 - - GACTCCGTGA AGGGACGATT CACCATCTCC CAAGGCAACA CCAAGAACAC AG -            #TGAATCTG    240                                                                 - - CAAATGAACA GCCTGACACC TGAGGACACG GCCATCTACA GTTGTGCGGC AA -            #CCAGTAGT    300                                                                 - - TTTTACTGGT ACTGCACCAC GGCGCCTTAT AACGTCTGGG GTCAGGGGAC CC -            #AGGTCACC    360                                                                 - - GTCTCCTCAC TAGTTACCCG TACGACGTTC CGGACTACGG TTCTTAATAG AA - #TTC             415                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:99:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 406 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:99:                       - - CTCGAGTCTG GGGGAGGCTC GGTGCAGGGT GGAGGGTCTC TGAGACTCTC CT -             #GTGCAATC     60                                                                 - - TCTGGATACA CGTACGGTAG CTTCTGTATG GGCTGGTTCC GCGAGGGTCC AG -            #GCAAGGAA    120                                                                 - - CGTGAGGGGA TCGCAACTAT TCTTAATGGT GGTACTAACA CATACTATGC CG -            #ACTCGGTG    180                                                                 - - AAGGGCCGAT TCACCATCTC CCAAGACAGC ACGTTGAAGA CGATGTATCT GC -            #TAATGAAC    240                                                                 - - AACCTGAAAC CTGAAGACAC GGGCACCTAT TACTGTGCTG CAGAACTAAG TG -            #GTGGTAGT    300                                                                 - - TGTGAATTGC CTTTGCTATT TGACTACTGG GGCCAGGGCA CCCAGGTCAC CG -            #TCTCCTCA    360                                                                 - - CTAGTTACCC GTACGACGTT CCGGACTACG GTTCTTAATA GAATTC   - #                    406                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:100:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 427 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:100:                      - - CTCGAGTCTG GGGGAGGCTC GGTGCAGGCT GGAGGGTCTC TGAGACTCTC CT -             #GTACAGGC     60                                                                 - - TCTGGATTCC CCTATAGTAC CTTCTGTCTG GGGTGGTTCC GCCAGGCTCC AG -            #GGAAGGAG    120                                                                 - - CGTGAGGGGG TCGCGGGTAT TAATAGTGCA GGAGGTAATA CTTACTATGC CG -            #ACGCCGTG    180                                                                 - - AAGGGCCGAT TCACCATCTC CCAAGGGAAT GCCAAGAATA CGGTGTTTCT GC -            #AAATGGAT    240                                                                 - - AACTTGAAAC CTGAGGACAC GGCCATCTAT TACTGCGCGG CGGATAGTCC AT -            #GTTACATG    300                                                                 - - CCGACTATGC CCGCTCCCCC GATACGAGAC AGTTTTGGCT GGGATGATTT TG -            #GCCAGGGG    360                                                                 - - ACCCAGGTCA CCGTCTCCTC ACTAGTTACC CGTACGACGT TCCGGACTAC GG -            #TTCTTAAT    420                                                                 - - AGAATTC                 - #                  - #                       - #         427                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:101:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 409 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:101:                      - - CTCGAGTCAG GGGGAGGCTC GGTACAGGTT GGAGGGTCTC TGAGACTCTC CT -            #GTGTAGCC     60                                                                 - - TCTACTCACA CCGACAGTAG CACCTGTATA GGCTGGTTCC GCCAGGCTCC AG -            #GGAAGGAG    120                                                                 - - CGCGAGGGGG TCGCAAGTAT ATATTTTGGT GATGGTGGTA CGAATTATCG CG -            #ACTCCGTG    180                                                                 - - AAGGGCCGAT TCACCATCTC CCAACTCAAC GCCCAGAACA CAGTGTATCT GC -            #AAATGAAC    240                                                                 - - AGCCTGAAAC CTGAGGACAG CGCCATGTAC TACTGTGCAA TCACTGAAAT TG -            #AGTGGTAT    300                                                                 - - GGGTGCAATT TAAGGACTAC TTTTACTCGC TGGGGCCAGG GGACCCAGGT CA -            #CCGTCTCC    360                                                                 - - TCACTAGTTA CCCGTACGAC GTTCCGGACT ACGGTTCTTA ATAGAATTC  - #                  409                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:102:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 445 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:102:                      - - CTCGAGTCTG GGGGAGGCTC GGTACAAACT GGAGGGTCTC TGAGACTCTC TT -             #GCGAAATC     60                                                                 - - TCTGGATTGA CTTTTGATGA TTCTGACGTG GGGTGGTACC GCCAGGCTCC AG -            #GGGATGAG    120                                                                 - - TGCAAATTGG TCTCAGGTAT TCTGAGTGAT GGTACTCCAT ATACAAAGAG TG -            #GAGACTAT    180                                                                 - - GCTGAGTCTG TGAGGGGCCG GGTTACCATC TCCAGAGACA ACGCCAAGAA CA -            #TGATATAC    240                                                                 - - CTTCAAATGA ACGACCTGAA ACCTGAGGAC ACGGCCATGT ATTACTGCGC GG -            #TAGATGGT    300                                                                 - - TGGACCCGGA AGGAAGGGGG AATCGGGTTA CCCTGGTCGG TCCAATGTGA AG -            #ATGGTTAT    360                                                                 - - AACTATTGGG GCCAGGGGAC CCAGGTCACC GTCTCCTCAC TAGTTACCCG TA -            #CGACGTTC    420                                                                 - - CGGACTACGG TTCTTAATAG AATTC          - #                  - #                  445                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:103:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 394 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:103:                      - - CTCGAGTCTG GAGGAGGCTC GGTGCAGGCT GGAGGGTCTC TGAGACTCTC CT -             #GTGTAGCC     60                                                                 - - TCTGGATTCA ATTTCGAAAC TTCTCGTATG GCGTGGTACC GCCAGACTCC AG -            #GAAATGTG    120                                                                 - - TGTGAGTTGG TCTCAAGTAT TTACAGTGAT GGCAAAACAT ACTACGTCGA CC -            #GCATGAAG    180                                                                 - - GGCCGATTCA CCATTTCTAG AGAGAATGCC AAGAATACAT TGTATCTACA AC -            #TGAGCGGC    240                                                                 - - CTCAAACCTG AGGACACGGC CATGTATTAC TGTGCGCCGG TTGAATATCC TA -            #TTGCAGAC    300                                                                 - - ATGTGTTCGA GATACGGCGA CCCGGGGACC CAGGTCACCG TCTCCTCACT AG -            #TTACCCGT    360                                                                 - - ACGACGAACC GGACTACGGT TCTTAATAGA ATTC       - #                  -     #       394                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:104:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 433 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:104:                      - - CTCGAGTCTG GGGGAGGCTC GGTGCAGGTT GGAGGGTCTC TGAAACTCTC CT -             #GTAAAATC     60                                                                 - - TCTGGAGGTA CCCCAGATCG TGTTCCTAAA TCTTTGGCCT GGTTCCGCCA GG -            #CTCCAGAG    120                                                                 - - AAGGAGCGCG AGGGGATCGC AGTTCTTTCG ACTAAGGATG GTAAGACATT CT -            #ATGCCGAC    180                                                                 - - TCCGTGAAGG GCCGATTCAC CATCTTCTTA GATAATGACA AGACCACTTT CT -            #CCTTACAA    240                                                                 - - CTTGATCGAC TGAACCCGGA GGACACTGCC GACTACTACT GCGCTGCAAA TC -            #AATTAGCT    300                                                                 - - GGTGGCTGGT ATTTGGACCC GAATTACTGG CTCTCTGTGG GTGCATATGC CA -            #TCTGGGGC    360                                                                 - - CAGGGGACCC AGGTCACCGT CTCCTCACTA GTTACCCGTA CGACGTTCCG GA -            #CTACGGTT    420                                                                 - - CTTAATAGAA TTC              - #                  - #                      - #     433                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:105:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 416 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:105:                      - - CAGGTGAAAC TGCTCGAGTC TGGGGGAGGC TCGGTGCAGG CTGGGGGGTC TC -             #TGACACTC     60                                                                 - - TCTTGTGTAT ACACCAACGA TACTGGGACC ATGGGATGGT TTCGCCAGGC TC -            #CAGGGAAA    120                                                                 - - GAGTGCGAAA GGGTCGCGCA TATTACGCCT GATGGTATGA CCTTCATTGA TG -            #AACCCGTG    180                                                                 - - AAGGGGCGAT TCACGATCTC CCGAGACAAC GCCCAGAAAA CGTTGTCTTT GC -            #GAATGAAT    240                                                                 - - AGTCTGAGGC CTGAGGACAC GGCCGTGTAT TACTGTGCGG CAGATTGGAA AT -            #ACTGGACT    300                                                                 - - TGTGGTGCCC AGACTGGAGG ATACTTCGGA CAGTGGGGTC AGGGGGCCCA GG -            #TCACCGTC    360                                                                 - - TCCTCACTAG CTAGTTACCC GTACGACGTT CCGGACTACG GTTCTTAATA GA - #ATTC            416                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:106:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 361 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:106:                      - - CTCGAGTCTG GGGGAGGCTC GGTCCAACCT GGAGGATCTC TGACACTCTC CT -             #GTACAGTT     60                                                                 - - TCTGGGGCCA CCTACAGTGA CTACAGTATT GGATGGATCC GCCAGGCTCC AG -            #GGAAGGAC    120                                                                 - - CGTGAAGTAG TCGCAGCCGC TAATACTGGT GCGACTAGTA AATTCTACGT CG -            #ACTTTGTG    180                                                                 - - AAGGGCCGAT TCACCATTTC CCAAGACAAC GCCAAGAATA CGGTATATCT GC -            #AAATGAGC    240                                                                 - - TTCCTGAAAC CTGAGGACAC GGCCATCTAT TACTGTGCGG CAGCGGACCC AA -            #GTATATAT    300                                                                 - - TATAGTATCC TCCATTGAGT ATAAGTACTG GGGCCAGGGG ACCCAGGTCA CC -            #GTCTCCTC    360                                                                 - - A                  - #                  - #                  - #                  361                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:107:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 354 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:107:                      - - CTCGAGTCAG GGGGAGGCTC GGTGGAGGCT GGAGGGTCTC TGAGACTCTC CT -             #GTACAGCC     60                                                                 - - TCTGGATACG TATCCTCTAT GGCCTGGTTC CGCCAGGTTC CAGGGCAGGA GC -            #GCGAGGGG    120                                                                 - - GTCGCGTTTG TTCAAACGGC TGACAATAGT GCATTATATG GCGACTCCGT GA -            #AGGGCCGA    180                                                                 - - TTCACCATCT CCCACGACAA CGCCAAGAAC ACGCTGTATC TGCAAATGCG CA -            #ACCTGCAA    240                                                                 - - CCTGACGACA CTGGCGTGTA CTACTGTGCG GCCCAAAAGA AGGATCGTAC TA -            #GATGGGCC    300                                                                 - - GAGCCTCGAG AATGGAACAA CTGGGGCCAG GGGACCCAGG TCACCGTCTC CT - #CA              354                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:108:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 381 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:108:                      - - CTCGAGTCAG GTGTCCGGTC TGATGTGCAG CTGGTGGCGT CTGGGGGAGG CT -             #CGGTGCAG     60                                                                 - - GCTGGAGGCT CTCTGAGACT CTCCTGTACA GCCTCTGGAG ACAGTTTCAG TA -            #GATTTGCC    120                                                                 - - ATGTCTTGGT TCCGCCAGGC TCCAGGGAAG GAGTGCGAAT TGGTCTCAAG CA -            #TTCAAAGT    180                                                                 - - AATGGAAGGA CAACTGAGGC CGATTCCGTG CAAGGCCGAT TCACCATCTC CC -            #GAGACAAT    240                                                                 - - TCCAGGAACA CAGTGTATCT GCAAATGAAC AGCCTGAAAC CCGAGGACAC GG -            #CCGTGTAT    300                                                                 - - TACTGTGGGG CAGTCTCCCT AATGGACCGA ATTTCCCAAC ATGGGTGCCG GG -            #GCCAGGGA    360                                                                 - - ACCCAGGTCA CCGTCTCCTT A           - #                  - #                     381                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:109:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:109:                      - -      Gly Gln Pro Arg Glu Pro Gln Val - # Tyr Thr Leu Pro Pro Ser        Arg Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Leu                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:110:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:110:                      - -      Gly Gln Pro Arg Glu Pro Gln Val - # Tyr Thr Leu Pro Pro Ser       Arg Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Met                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:111:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:111:                      - -      Gly Gln Pro Arg Glu Pro Gln Val - # Tyr Thr Leu Pro Pro Ser       Gln Glu                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Met                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:112:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:112:                      - -      Leu Leu Gly Gly Pro Ser Val Phe - # Leu Phe Pro Pro Lys Pro       Lys Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Met Ile Ser Arg Thr Pro                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:113:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:113:                      - -      Val Ala Gly Pro Ser Val Phe Leu - # Phe Pro Pro Lys Pro Lys       Asp Thr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Leu Met Ile Ser Arg Thr Pro                                                          20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:114:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:114:                      - -      Phe Leu Gly Gly Pro Ser Val Phe - # Leu Phe Pro Pro Lys Pro       Lys Asp                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Thr Leu Met Ile Ser Arg Thr Pro                                                      20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:115:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:115:                      - -      Lys Gly Gln Pro Arg Glu Pro Gln - # Val Tyr Thr Leu Pro Pro       Ser Arg                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Asp Glu Leu                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:116:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:116:                      - -      Lys Gly Gln Pro Arg Glu Pro Gln - # Val Tyr Thr Leu Pro Pro       Ser Arg                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Glu Met                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:117:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:117:                      - -      Lys Gly Gln Pro Arg Glu Pro Gln - # Val Tyr Thr Leu Pro Pro       Ser Gln                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Glu Glu Met                                                          - -  - - (2) INFORMATION FOR SEQ ID NO:118:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:118:                      - -      Glu Val Lys Leu Val Glu Ser Gly - # Gly Gly Leu Val Glu Pro       Gly Gly                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Ser Leu Arg Leu Ser Cys Ala Thr - # Ser Gly Phe Thr Phe Ser                         20 - #                 25 - #                 30              - -  - - (2) INFORMATION FOR SEQ ID NO:119:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:119:                      - -      Glu Val Gln Leu Leu Ser Gly Gly - # Gly Leu Val Gln Pro Gly        Gly Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Leu Arg Leu Ser Cys Ala Ala Ser - # Gly Phe Thr Phe Ser                             20 - #                 25                                     - -  - - (2) INFORMATION FOR SEQ ID NO:120:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:120:                      - -      Trp Gly Gln Gly Thr Thr Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:121:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:121:                      - -      Trp Gly Gln Gly Thr Met Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:122:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:122:                      - -      Trp Gly Gln Gly Thr Thr Leu Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:123:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:123:                      - -      Trp Gly Gln Gly Thr Ser Val Thr - # Val Ser Ala                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:124:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:124:                      - -      Trp Gly Ala Gly Thr Thr Val Thr - # Val Ser Ser                          1             - #  5                - #   10                              - -  - - (2) INFORMATION FOR SEQ ID NO:125:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:125:                      - -      Asp Tyr Tyr Gly Ser Ser Tyr Phe - # Asp Val Trp Gly Ala Gly        Thr Thr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Val Thr Val Ser Ser                                                                  20                                                            - -  - - (2) INFORMATION FOR SEQ ID NO:126:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:126:                      - -      Lys Val Asp Lys Arg Val Glu Leu - # Lys Thr Pro Leu Gly Asp       Thr Thr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      His Thr Cys Pro Arg Cys Pro Glu - # Pro Lys Cys Ser Asp Thr       Pro Pro                                                                                          20 - #                 25 - #                 30             - -      Pro Cys Pro Arg Cys Pro Glu Pro - # Lys Ser Cys Asp Thr Pro        Pro Pro                                                                                      35     - #             40     - #             45                  - -      Cys Pro Arg Cys Pro Ala Pro Glu - # Leu Leu Gly Gly Pro Ser       Val Phe                                                                                  50         - #         55         - #         60                      - -      Leu Phe Pro                                                              65                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:127:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:127:                      - -      Lys Val Asp Lys Lys Ala Glu Pro - # Lys Ser Cys Asp Lys Thr       His Thr                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Cys Pro Pro Cys Pro Ala Pro Glu - # Leu Leu Gly Gly Pro Ser       Val Phe                                                                                          20 - #                 25 - #                 30             - -      Leu Phe Pro                                                                      35                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:128:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:128:                      - -      Lys Val Lys Val Thr Val Glu Arg - # Lys Cys Cys Val Glu Cys        Pro Pro                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Cys Pro Ala Pro Pro Val Ala Gly - # Pro Ser Val Phe Leu Phe       Pro                                                                                              20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:129:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:129:                      - -      Lys Val Asp Lys Arg Val Glu Ser - # Lys Tyr Gly Pro Pro Cys        Pro Ser                                                                              1             - #  5                - #   10               - #         15                                                                               - -      Cys Pro Ala Pro Glu Phe Leu Gly - # Gly Pro Ser Val Phe Leu       Phe Pro                                                                                          20 - #                 25 - #                 30             - -  - - (2) INFORMATION FOR SEQ ID NO:130:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -         (xi) SEQUENCE DESCRIPTION: SEQ - #ID NO:130:                      - -      Trp Gly Arg Gly Thr Leu Val Thr - # Val Ser Ser                          1             - #  5                - #   10                            __________________________________________________________________________

We claim:
 1. An immunoglobulin comprising two heavy polypeptide chains,each heavy chain containing a binding site specific for an antigen, saidimmunoglobulin containing a variable (V_(HH)) region and a constantregion, said constant region being devoid of fist constant domain C_(H)1, wherein the immunoglobulin is devoid of polypeptide light chains. 2.The immunoglobulin according to claim 1, wherein each variable region ofeach heavy chain polypeptide contains binding site specific for anantigen.
 3. The immunoglobulin according to claim 1, which is a type Gimmunoglobulin of class 2 (IgG2) or it is a type G immunoglobulin ofclass 3 (IgG3).
 4. The immunoglobulin according to claim 1, asobtainable by purification from the serum of Camelids, wherein saidimmunoglobulin or fragment:is not adsorbed by affinity chromatography ona Protein G column, is adsorbed by affinity chromatography on a ProteinA column, has a molecular weight of around 100 Kd after elution with apH 4.5 buffer (0.15 M NaCl, 0.58% acetic acid adjusted to pH 4.5 byNaOH), consists of heavy γ2 polypeptide chains of a molecular weight ofaround 45 Kd preferably 46 Kd after reduction.
 5. The immunoglobulinaccording to claim 1, obtainable by purification from serum of Camelids,wherein the immunoglobulin:is adsorbed by affinity chromatography on aProtein A column; has a molecular weight of about 100 Kd after elutionwith a pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid); is adsorbed byaffinity chromatography on a Protein G column and eluted with pH 3.5buffer (0.15 M NaCl, 0.58% acetic acid); and comprises heavy γ3polypeptide chains having a molecular weight between 43 and 47 Kd afterreduction.
 6. The immunoglobulin according to claim 5, which comprisesheavy γ3 polypeptide chains having a molecular weight of about 45 Kd,after reduction.
 7. A fragment of the immunoglobulin according to claim1, wherein said fragment isa fragment corresponding to one heavypolypeptide chain of an immunoglobulin devoid of light chains, whereinthe amino acid residue at position 45 in the framework 2 region of theVHH domain is selected from the group consisting of charged amino acids,and a cysteine residue and wherein the heavy polypeptide chain containsan antigen binding site specific for an antigen.
 8. A fragment of atleast 10 amino acid residues of the variable region of a heavypolypeptide chain of the immunoglobulin according to claim 1, containingan amino acid residue corresponding to position 45 of said heavy chain,wherein said amino acid residue at position 45 is selected from thegroup consisting of charged amino acids, and a cysteine residue, whereinsaid fragment comprises a binding site specific for an antigen.
 9. Afragment of at least 10 amino acid residues of the variable region of aheavy polypeptide chain of the immunoglobulin according to claim 1,containing an amino acid residue corresponding to position 45 of saidheavy chain, wherein said amino acid residue at position 45 is not aleucine residue, wherein said fragment comprises a binding site specificfor an antigen.
 10. A fragment of the immunoglobulin according to claim1, wherein said fragment comprises a binding site specific for anantigen and is selected from the following group:fragments obtained bypartial digestion of the immunoglobulin of claim 1 with papain leadingto the Fc fragment (constant fragment) and leading to F_(HH) h fragment(containing the antigen binding sites of the heavy chains) or its dimerF(V_(HH) h)₂, or a fragment obtained by further digestion with papain ofthe Fc fragment, leading to the Fc' fragment corresponding to theC-terminal part of the Fc fragment; and homologous fragments obtainedwith other proteolytic enzymes.
 11. A fragment of the variable region ofthe heavy polypeptide chain of the immunoglobulin according to claim 1,which comprises a CDR3 domain and includes the amino acid residuecorresponding to position 45 of said heavy polypeptide chain whereinsaid amino acid residue is selected from the group consisting of chargedamino acids, and a cysteine residue, wherein said fragment comprises abinding site specific for an antigen.
 12. The variable region (V_(HH))of the immunoglobulin according to claim
 1. 13. The fragment accordingto any one of claims 7, or 8 to 12, wherein each variable region of eachheavy chain polypeptide contains binding site specific for an antigen.14. The fragment according to any one of claims 7, or 8 to 12, which isa type G immunoglobulin of class 2 (IgG2) or it is a type Gimmunoglobulin of class 3 (IgG3).